Crystalline forms of 5-chloro-N4-[-2-(dimethylphosphoryl) phenyl]-N2-{2-methoxy-4-[4-(4-methylpiperazin-1-yl) piperidin-1-yl] phenyl} pyrimidine-2,4-diamine

ABSTRACT

Novel crystalline forms of brigatinib, pharmaceutical compositions comprising the same, and methods of their preparation and use of the same are disclosed herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/429,988, filed Jun. 3, 2019, which is a division of U.S. patentapplication Ser. No. 15/520,694, filed Sep. 25, 2017, now patent Ser.No. 10/385,078, which is a 35 U.S.C. § 371 United States National PhaseApplication of, and claims priority to, PCT Application No.PCT/US2015/056701, filed Oct. 21, 2015, which claims priority under 35U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/066,849,filed Oct. 21, 2014. The entire contents of the aforesaid applicationsare incorporated by reference herein in its entirety.

This application is directed to novel crystalline forms of5-chloro-N4-[2-(dimethylphosphoryl)phenyl]-N2-{2-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]pyrimidine-2,4-diamine(also referred to as, “AP26113” and “brigatinib”), compositionscomprising such crystalline forms, and methods of their preparation anduse.

Brigatinib has the chemical formula C₂₉H₃₉ClN₇O₂P which corresponds to aformula weight of 584.09 g/mol. Its chemical structure is shown below:

Brigatinib is a multi-targeted tyrosine-kinase inhibitor useful for thetreatment of non-small cell lung cancer (NSCLC) and other diseases. Itis a potent inhibitor of ALK (anaplastic lymphoma kinase) and is inclinical development for the treatment of adult patients with ALK-drivenNSCLC. Crizotinib (XALKORI®) is an FDA approved drug for first-linetreatment of ALK-positive NSCLC. “Despite initial responses tocrizotinib, the majority of patients have a relapse within 12 months,owing to the development of resistance.” Shaw et al., New Eng. J. Med.370:1189-97 2014. Thus, a growing population of cancer patients are inneed of new and effective therapies for ALK-positive cancers.

Brigatinib is also potentially useful for treating other diseases orconditions in which ALK or other protein kinases inhibited by brigatinibare implicated. Such kinases and their associated disorders orconditions are disclosed in WO 2009/143389, both of which are herebyincorporated herein by reference for all purposes.

Knowledge of the potential polymorphic forms of active pharmaceuticalingredients (API) such as brigatinib can be useful in the development ofdrugs, as is knowledge of characteristics of those polymorphs. Notknowing the specific polymorphic form present or desired in the API canresult in inconsistent manufacturing of the API, thus results with thedrug can potentially vary between various lots of the API. In addition,knowledge of the polymorphic forms of an API informs and permits longterm systematic stability determination of the API. Once a specificpolymorphic form is selected for pharmaceutical development, a methodfor reproducibly preparing that polymorphic form can be useful. It isalso useful for there to be a process for making APIs such as brigatinibat or above a specified level of chemical and/or polymorphic purity.

The chemical structure of brigatinib was first disclosed in WO2009/143389, which is also owned by Applicant (ARIAD Pharmaceuticals,Inc.) and is hereby incorporated herein by reference in its entirety forall purposes. Example 122 of WO 2009/143389 discloses the synthesis ofbrigatinib and states that the product was obtained as an off-whitesolid but does not provide further characterization, such as chemicalpurity or solid form. Example 122 does not state to what degree, if any,its product was crystalline.

Provided herein are certain crystalline and other polymorphic forms ofbrigatinib, certain of which are suitable for pharmaceutical formulationdevelopment.

In some embodiments, the present disclosure relates to crystallinebrigatinib. In some embodiments, the present disclosure relates tosubstantially pure crystalline brigatinib.

In one embodiment, the present disclosure is directed to polymorphs ofbrigatinib. The polymorphs of brigatinib are herein designated as FormA, Form B, Form C, Form D, Form E, Form F, Form G, Form H, Form J, andForm K.

In another embodiment, the present disclosure is directed tosubstantially pure crystalline forms of brigatinib. The substantiallypure crystalline forms of brigatinib are herein designated as Form A,Form B, Form C, Form D, Form E, Form F, Form G, Form H, Form J, and FormK.

In another embodiment, the present disclosure is directed topharmaceutical compositions consisting essentially of a crystalline formof brigatinib disclosed herein and at least one additional componentchosen from pharmaceutically acceptable carriers, pharmaceuticallyacceptable vehicles, and pharmaceutically acceptable excipients. Inanother embodiment, the present disclosure is directed to pharmaceuticalcompositions consisting of at least one polymorph of brigatinibdisclosed herein and at least one additional component chosen frompharmaceutically acceptable carriers, pharmaceutically acceptablevehicles, and pharmaceutically acceptable excipients.

In another embodiment, the present disclosure is directed to a method oftreating a disorder and/or condition in a subject that responds to theinhibition of a protein kinase by administering to the subject apolymorph of brigatinib disclosed herein. In certain embodiments, atleast one of the disorders and/or conditions is non-small cell lungcancer (NSCLC).

In another embodiment, the present disclosure is directed to a method oftreating a disorder and/or condition in a subject that responds to theinhibition of a protein kinase by administering to the subject asubstantially pure crystalline form of brigatinib disclosed herein. Incertain embodiments, at least one of the disorders and/or conditions isNSCLC when the protein kinase is ALK or a mutant form thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain embodiments of the presentdisclosure. The disclosure may be understood by reference to one or moreof these drawings in combination with the detailed description ofembodiments disclosed herein.

FIG. 1 is a synthetic scheme for brigatinib.

FIG. 2 is an X-Ray Powder Diffraction (XRPD) pattern obtained from asample of brigatinib Form A. Relative Intensity (in counts) is shown onthe vertical axis and angle (in degrees two theta (°2θ)) is shown on thehorizontal axis.

FIG. 3 is a sorption-desorption plot of the dynamic vapor sorption (DVS)experiment of a sample of brigatinib Form A. Change in mass (%) is shownon the vertical axis and Target RH (%) is shown on the horizontal axis.

FIG. 4 is a differential scanning calorimetry (DSC) scan obtained from asample of Form A of brigatinib. Heat flow (mW) is shown on the verticalaxis and temperature (° C.) is shown on the horizontal axis.

FIG. 5A is a thermogravimetric analysis/single differential thermalanalysis thermogram (TGA/SDTA) for a sample of brigatinib Form A.

FIG. 5B is a thermogravimetric mass spectrometry (TGMS) thermogram for asample of brigatinib Form A.

FIG. 6 is an ¹H-NMR spectrum obtained for a sample of brigatinibdissolved in CD₃OD. Normalized intensity is shown on the vertical axisand chemical shift (ppm) is shown on the horizontal axis.

FIG. 7 is a ¹³C-NMR spectrum obtained for a sample of brigatinibdissolved in CDCl₃. Normalized intensity is shown on the vertical axisand chemical shift (ppm) is shown on the horizontal axis.

FIG. 8 is a mass spectral fragmentation pattern of a sample ofbrigatinib Form A. Relative abundance is shown on the vertical axis andatomic weight (m/z) is shown on the horizontal axis.

FIGS. 9A-9E depict the fragmentation pattern of ions of a sample ofbrigatinib Form A using collisional activation, measured using anelectrospray time of flight mass spectrometer. Relative abundance isshown on the vertical axis and atomic weight (m/z) is shown on thehorizontal axis.

FIG. 10 is a crystal structure of brigatinib Form A, as determined bysingle-crystal X-Ray diffraction.

FIG. 11 is a differential scanning calorimetry (DSC) scan obtained froma sample of brigatinib Form B. Heat flow (mW) is shown on the verticalaxis and temperature (° C.) is shown on the horizontal axis.

FIG. 12 is a cyclic differential scanning calorimetry (DSC) scan of asample of brigatinib Form B; heating to 190° C. at 10° C./min andcooling to 25° C. at the same rate. Heat flow (mW) is shown on thevertical axis and temperature (° C.) is shown on the horizontal axis.

FIG. 13A is a cyclic differential scanning calorimetry (DSC) scan of asample of brigatinib Form B; heating to 190° C. at 10° C./min, coolingto 25° C. at the same rate, followed by a second heating to 300° C. atthe same rate. Heat flow (mW) is plotted on the vertical axis andtemperature (° C.) is plotted on the horizontal axis.

FIG. 13B is a cyclic differential scanning calorimetry (DSC) scan of asample of brigatinib Form B; heating to 190° C. at 10° C./min, coolingto 25° C. at the same rate, followed by a second heating to 300° C. atthe same rate. Heat flow (mW) is plotted on the vertical axis and time(min) is plotted on the horizontal axis.

FIG. 13C is a thermogravimetric analysis/single differential thermalanalysis thermogram (TGA/SDTA) for a sample of brigatinib Form B.

FIG. 13D is a thermogravimetric mass spectrometry (TGMS) thermogram fora sample of brigatinib Form B.

FIG. 14 is a X-Ray Powder Diffraction (XRPD) pattern obtained from asample of brigatinib Form B. Relative Intensity (in counts) is shown onthe vertical axis and the angle (in degrees two theta (°2θ)) is shown onthe horizontal axis.

FIG. 15 is a differential scanning calorimetry (DSC) scan obtained froma sample of brigatinib Form C. Heat flow (mW) is shown on the verticalaxis and temperature (° C.) is shown on the horizontal axis.

FIG. 16A is a thermogravimetric analysis/single differential thermalanalysis (TGA/SDTA) thermogram of a sample of brigatinib Form C. A watermass loss of 4.25% was observed up to about 75° C., corresponding to1.44 water molecules.

FIG. 16B is a thermogravimetric mass spectrometry (TGMS) thermogram of asample of brigatinib Form C. A water mass loss of 4.25% was observed upto about 75° C., corresponding to 1.44 water molecules.

FIG. 17A is a thermogravimetric analysis/single differential thermalanalysis (TGA/SDTA) thermogram of a sample of brigatinib Form C. A watermass loss of 6.14% was observed up to about 75° C., corresponding to2.12 water molecules.

FIG. 17B is a thermogravimetric mass spectrometry (TGMS) thermogram of asample of brigatinib Form C. A water mass loss of 6.14% was observed upto about 75° C., corresponding to 2.12 water molecules.

FIG. 18 is a X-Ray Powder Diffraction (XRPD) pattern obtained from asample of brigatinib Form C. Relative Intensity (in counts) is shown onthe vertical axis and the angle (in degrees two theta (°2θ)) is shown onthe horizontal axis.

FIG. 19 is a X-Ray Powder Diffraction (XRPD) pattern obtained from asample of brigatinib Form D. Relative Intensity (in counts) is shown onthe vertical axis and the angle (in degrees two theta (°2θ)) is shown onthe horizontal axis.

FIG. 19A is a thermogravimetric analysis/single differential thermalanalysis thermogram (TGA/SDTA) for a sample of brigatinib Form D.

FIG. 19B is a thermogravimetric mass spectrometry (TGMS) thermogram fora sample of brigatinib Form D.

FIG. 20A is a thermogravimetric analysis/single differential thermalanalysis (TGA/SDTA) of a sample of brigatinib Form E.

FIG. 20B is a thermogravimetric mass spectrometry (TGMS) thermogram of asample of brigatinib Form E.

FIG. 21 is a X-Ray Powder Diffraction (XRPD) pattern obtained from asample of brigatinib Form E. Relative Intensity (in counts) is shown onthe vertical axis and the angle (in degrees two theta (°2θ)) is shown onthe horizontal axis.

FIG. 22 is a thermogravimetric analysis/single differential thermalanalysis (TGA/SDTA) of a sample of brigatinib Form F.

FIG. 23 is a X-Ray Powder Diffraction (XRPD) pattern obtained from asample of brigatinib Form F. Relative Intensity (in counts) is shown onthe vertical axis and the angle (in degrees two theta (°2θ)) is shown onthe horizontal axis.

FIG. 24 is a X-Ray Powder Diffraction (XRPD) pattern obtained from asample of brigatinib Form G. Relative Intensity (in counts) is shown onthe vertical axis and the angle (in degrees two theta (°2θ)) is shown onthe horizontal axis.

FIG. 25 is a X-Ray Powder Diffraction (XRPD) pattern obtained from asample of brigatinib Form H. Relative Intensity (in counts) is shown onthe vertical axis and the angle (in degrees two theta (°2θ)) is shown onthe horizontal axis.

FIG. 26 is a X-Ray Powder Diffraction (XRPD) pattern obtained from themixture of a sample of brigatinib Form A and Form J. Relative Intensity(in counts) is shown on the vertical axis and the angle (in degrees twotheta (°2θ)) is shown on the horizontal axis.

FIG. 27A is a X-Ray Powder Diffraction (XRPD) overlay pattern obtainedfrom a sample of a mixture of brigatinib Form A and Form K, a sample ofa mixture of brigatinib Form A and Form L, and a sample of brigatinibForm A. Relative Intensity (in counts) is shown on the vertical axis andthe angle (in degrees two theta (°2θ)) is shown on the horizontal axis.

FIG. 27B is an expansion of FIG. 27A.

FIG. 28 contains overlaid X-Ray Powder Diffraction (XRPD) patterns ofbrigatinib Form A that has been subjected to grinding experiments forvarious lengths. Relative intensity (in counts) is shown on the verticalaxis and the angle (in degrees two theta (°2θ)) is shown on thehorizontal axis.

FIG. 29 depicts the solubility data for brigatinib Form A and Form B at25° C. and 37° C., at varying pH values.

FIG. 30A is an expansion graph of FIG. 30B, showing the concentrationsof brigatinib Forms A and B vs. time obtained from the intrinsicdissolution rate (IDR) experiments, where concentration (mg/mL) isplotted on the vertical axis and time (min) is plotted on the horizontalaxis.

FIG. 30B is a plot of the concentrations of brigatinib Forms A and B vs.time obtained from the intrinsic dissolution rate (IDR) experiments,where concentration (mg/mL) is plotted on the vertical axis and time(min) is plotted on the horizontal axis.

FIG. 31 is a plot of concentration of brigatinib Forms A and B vs. timeobtained from the IDR experiments at 25° C. and 37° C. in pH 1.0 HClbuffer. Concentration (mg/mL) is shown on the vertical axis and time(min) is shown on the horizontal axis.

FIG. 32A is an expansion graph of FIG. 32B, showing the plot of theconcentration of brigatinib Form A and Form B vs. time obtained from theIDR experiments at 25° C. and 37° C. in pH 6.5 buffer. Concentration(mg/mL) is plotted on the vertical axis and time (min) is plotted on thehorizontal axis.

FIG. 32B is a plot of the concentration of brigatinib Form A and Form Bvs. time obtained from the IDR experiments at 25° C. and 37° C. in pH6.5 buffer. Concentration (mg/mL) is plotted on the vertical axis andtime (min) is plotted on the horizontal axis.

FIG. 33A is an expansion graph of FIG. 33B, showing the concentration ofbrigatinib Form A and Form B vs. time obtained from the IDR experimentsat 25° C. and 37° C. in SGF. Concentration (mg/mL) is plotted on thevertical axis and time (min) is plotted on the horizontal axis.

FIG. 33B is a plot of the concentration of brigatinib Form A and Form Bvs. time obtained from the IDR experiments at 25° C. and 37° C. in SGF.Concentration (mg/mL) is plotted on the vertical axis and time (min) isplotted on the horizontal axis.

FIG. 34 is a plot of the concentration of brigatinib Form A vs. timeobtained from the IDR experiments at 25° C. in water and aqueous buffersof pH 1.0, 4.5 and 6.5.

FIG. 35 is a plot of the concentration of brigatinib Form A vs. timeobtained from the IDR experiments at 37° C. in water and aqueous buffersof pH 1.0, 4.5 and 6.5.

FIG. 36 is a plot of the concentration of brigatinib Form B vs. timeobtained from the IDR experiments at 25° C. in water and aqueous buffersof pH 1.0, 4.5 and 6.5.

FIG. 37 is a plot of the concentration of brigatinib Form B vs. timeobtained from the IDR experiments at 37° C. in water and aqueous buffersof pH 1.0, 4.5 and 6.5.

FIG. 38A is an expansion graph of FIG. 38B, showing the concentration ofbrigatinib Form A vs. time obtained from the IDR experiments in water at25° C. and 37° C. Concentration (mg/mL) is plotted on the vertical axisand time (min) is plotted on the horizontal axis.

FIG. 38B is a plot of the concentration of brigatinib Form A vs. timeobtained from the IDR experiments in water at 25° C. and 37° C.Concentration (mg/mL) is plotted on the vertical axis and time (min) isplotted on the horizontal axis.

FIG. 39A is an expansion graph of FIG. 39B, showing the concentration ofbrigatinib Form A vs. time obtained from the IDR experiments in pH 6.5buffer at 25° C. and 37° C. Concentration (mg/mL) is plotted on thevertical axis and time (min) is plotted on the horizontal axis.

FIG. 39B is a plot of the concentration of brigatinib Form A vs. timeobtained from the IDR experiments in pH 6.5 buffer at 25° C. and 37° C.Concentration (mg/mL) is plotted on the vertical axis and time (min) isplotted on the horizontal axis.

FIG. 40A is an expansion graph of FIG. 40B, showing the concentration ofbrigatinib Form B vs. time obtained from the IDR experiments in pH 6.5buffer at 25° C. and 37° C. Concentration (mg/mL) is plotted on thevertical axis and time (min) is plotted on the horizontal axis.

FIG. 40B is a plot of the concentration of brigatinib Form B vs. timeobtained from the IDR experiments in pH 6.5 buffer at 25° C. and 37° C.Concentration (mg/mL) is plotted on the vertical axis and time (min) isplotted on the horizontal axis.

FIG. 41A is an expansion graph of FIG. 41B, showing the concentration ofbrigatinib Form A and Form B vs. time obtained from the IDR experimentsat 25° C. in pH 6.5 buffer. Concentration (mg/mL) is plotted on thevertical axis and time (min) is plotted on the horizontal axis.

FIG. 41B is a plot of the concentration of brigatinib Form A and Form Bvs. time obtained from the IDR experiments at 25° C. in pH 6.5 buffer.Concentration (mg/mL) is plotted on the vertical axis and time (min) isplotted on the horizontal axis.

FIG. 42A is an expansion graph of FIG. 42B, showing the concentration ofbrigatinib Form A and Form B vs. time obtained from the IDR experimentsat 37° C. in pH 6.5 buffer. Concentration (mg/mL) is plotted on thevertical axis and time (min) is plotted on the horizontal axis.

FIG. 42B is a plot of the concentration of brigatinib Form A and Form Bvs. time obtained from the IDR experiments at 37° C. in pH 6.5 buffer.Concentration (mg/mL) is plotted on the vertical axis and time (min) isplotted on the horizontal axis.

FIG. 43A is a plot of the concentration of brigatinib Form A and Form Bvs. time obtained from the dissolution rate (IDR) experiment in water at37° C. Concentration (mg/mL) is plotted on the vertical axis and time(min) is plotted on the horizontal axis.

FIG. 43B is an expansion graph of FIG. 43A, showing the concentration ofbrigatinib Form A and Form B vs. time obtained from the dissolution rate(IDR) experiment in water at 37° C. Concentration (mg/mL) is plotted onthe vertical axis and time (min) is plotted on the horizontal axis.

FIG. 44 is a DVS plot of Form B, wherein the total gain in mass at 95%RH corresponded to 2.26 water molecules.

FIG. 45 is a DVS plot of Form B, wherein the total gain in mass at 85%RH corresponded to 5.6 water molecules.

FIG. 46 is a DVS plot of Form B, wherein the total gain in mass at 95%RH corresponded to 5.15 water molecules.

FIG. 47 is a DVS plot of Form B, wherein the total gain in mass at 95%RH corresponded to 7.2 water molecules.

FIG. 48 is an overlay of XRPD patterns of Forms A, B, C and D.

FIG. 49 is an overlay of XRPD patterns of Forms A, B, C, D, E, F, G, H,and A mixed with J.

FIG. 50 is an inter-conversion scheme for Forms A, B, C and D based onexperiments. The dashed box shows that, at 30° C., increasing thehumidity lead to hydration of Form B to Form C and eventually to Form D.The changes are reversible upon humidity decrease. The solid-line boxshows that, at ambient humidity, increasing the temperature lead todehydration of Form C and Form D to Form B (at about 40° C.) and to FormA via solid-solid transition at about 150° C. These conversions are notreversible: Form A remains stable upon temperature decrease

Disclosed herein are various crystalline forms of brigatinib. As usedherein, the terms “crystalline form,” “polymorphic form,” and“polymorph” are used interchangeably, and refer to a solid form ofbrigatinib that is distinct from the amorphous form of brigatinib andfrom other solid form(s) of brigatinib as evidenced by certainproperties such as, for example, kinetic and/or thermodynamic stability,certain physical parameters, X-ray crystal structure, DSC, and/orpreparation processes. Polymorphic forms of a compound can havedifferent chemical and/or physical properties, including, for example,stabilities, solubilities, dissolution rates, optical properties,melting points, chemical reactivities, mechanical properties, vaporpressures, and/or densities. These properties can affect, for example,the ability to process and/or manufacture the drug substance and thedrug product, stability, dissolution, and/or bioavailability. Thus,polymorphism may affect at least one property of a drug including, butnot limited to, quality, safety, and/or efficacy.

While polymorphism classically refers to the ability of a compound tocrystallize into more than one crystalline form (having identicalchemical structure), the term “pseudopolymorphism” is typically appliedto solvate and hydrate crystalline forms. For purposes of thisdisclosure, however, both true polymorphs as well as pseudopolymorphs(i.e., hydrate and solvate forms) are included in the scope of the term“crystalline forms” and “polymorphic forms.” In addition, “amorphous”refers to a non-crystalline solid state.

It should be there can be variation in the angle of peaks (XRPD maximavalues) in XRPD diffractograms. Those of ordinary skill in the art areaware that a variance in 2-θ peak position may be observed, such as forexample a variance of ±0.2 °2θ or a variance of ±0.3 °2θ. Furthermore,those of ordinary skill in the art would recognize that the relativeintensities (expressed in counts) of peaks can vary between samples, forexample, due to preferred orientation. See, e.g., U.S. Pharmacopeia<941> X-Ray Diffraction. Accordingly, crystalline forms disclosed hereinhave X-ray powder diffraction patterns substantially as shown in certainfigures, e.g., Forms A-H respectively have X-ray powder diffractionpatterns substantially as shown in the FIGS. 2, 14, 18, 19, and 21-25.Of course, those of ordinary skill in the art would recognize that anyadditional component(s) in an XRPD sample can give contribute peaks tothe XRPD pattern observed for the sample which peaks can mask or overlap(either partially or completely) peaks attributable to the crystallineform(s) of brigatinib in the XRPD sample.

As used herein, the terms “isolated” and “substantially pure” mean thatmore than 50%, such as more than 60%, such as more than 70%, such asmore than 80%, such as more than 85%, such as more than 90%, such asmore than 95%, such as more than 99%, such as more than 99.5%, such asmore than 99.8%, or such as more than 99.9% of the brigatinib present ina sample is of a single crystalline form (as can be determined by amethod in accordance with the art). For example, some embodiments of theinvention is substantially pure crystalline brigatinib Form A. In someembodiments, the substantially pure crystalline form of brigatinibcontains less than 5%, such as less than 1%, such as less than 0.5%,such as less than 0.2%, or such as less than 0.1% of any other solidform of brigatinib (as can be determined by a method in accordance withthe art, such as XPRD analysis, for example).

As used herein, when used with reference to the chemical purity of acompound such as brigatinib, “pure” means that more than 90%, such asmore than 95%, such as more than 99%, such as more than 99.5%, such asmore than 99.8%, or such as more than 99.9% of the sum of allchemical(s) present in the selected material, e.g., in a sample of API,is the brigatinib molecule (as can be determined by a method inaccordance with the art).

The following abbreviations for solvents may be used herein:

-   -   DCM Dichloromethane    -   DMA N,N-Dimethylacetamide    -   DMF N,N-Dimethylformamide    -   DMSO Dimethylsulfoxide    -   EtOAc Ethyl acetate    -   EtOH Ethanol    -   IPA Isopropyl alcohol    -   LiHDMS lithium bis(trimethylsilyl)amide    -   MeCN Acetonitrile    -   MeOH Methanol    -   NMP N-Methylpyrrolidine    -   TFE 2,2,2-Trifluoroethanol    -   THF Tetrahydrofuran    -   2-methylTHF 2-Methyltetrahydrofuran

Other abbreviations (alphabetical order) that may be used hereininclude:

-   -   Am Amorphous    -   API Active Pharmaceutical Ingredient    -   AS Anti-solvent    -   DSC Differential Scanning Calorimetry    -   DVS Dynamic Vapor Sorption    -   HPLC High-Performance Liquid Chromatography    -   IDR Intrinsic Dissolution Rate    -   MS Mass Spectroscopy    -   NSCLC Non-Small Cell Lung Cancer    -   psi pounds per square inch    -   QSA Quantitative Solubility Assessment    -   RH Relative Humidity    -   S Solvent    -   SDTA Single Differential Thermal Analysis    -   SGF Simulated Gastric Fluid    -   SM Starting Material    -   TGA Thermogravimetric Analysis    -   TGMS Thermogravimetric Analysis Coupled with Mass Spectroscopy    -   VH-XRPD Variable humidity X-Ray Powder Diffraction    -   VT-XRPD Variable temperature X-Ray Powder Diffraction    -   Xantphos 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene    -   XRPD X-Ray Powder Diffraction

A “subject” to which/whom administration is contemplated includes, butis not limited to, a human (i.e., a male or female of any age group,e.g., a pediatric subject (e.g., infant, child, adolescent) or adultsubject (e.g., young adult, middle-aged adult or senior adult)), anotherprimate (e.g., cynomolgus monkeys, rhesus monkeys), a mammal, including,but is not limited to, cattle, pigs, horses, sheep, goats, cats, and/ordogs; and/or birds, including, but is not limited to, chickens, ducks,geese, quail, and/or turkeys.

XRPD patterns disclosed herein were obtained using the Crystallics T2high-throughput XRPD set-up. The plates were mounted on a Bruker GADDSdiffractometer equipped with a Hi-Star area detector. The XRPD platformwas calibrated using Silver Behenate for the long d-spacings andCorundum for the short d-spacings.

Data collection was carried out at room temperature using monochromaticCuKα radiation in the 2θ region between 1.5° and 41.5°. The diffractionpattern of each well was collected in two 2θ ranges (1.5°≤2θ≤21.5° forthe first frame, and 19.5°≤2θ≤41.5° for the second) with an exposuretime of 90 seconds for each frame. No background subtraction or curvesmoothing was applied to the XRPD patterns in the Figures.

The carrier material used during XRPD analysis was transparent toX-rays.

High resolution X-ray powder diffraction patterns disclosed herein werecollected on a D8 Advance system in the Brag-Brentano geometry equippedwith LynxEye solid state detector. The radiation used for collecting thedata was CuKα1 (λ=1.54056 Å) monochromatized by germanium crystal. Thepatterns were collected in the range of 4-41.5°2θ, with a step in therange of 0.016 °2θ without further processing. All patterns were takenat room temperature, approximately 295 K. The material was placed in aboron glass capillary of 0.3 mm diameter. For variable humidity andvariable temperature experiments disclosed herein, an ANSYCO HT chamberwas used. The material was placed on a fixed sample holder that wasmounted inside the chamber. The humidity was applied locally and variedfrom 10% to 80% (dew point). The temperature variation rate was 10°C./min.

The step used during the experiments were 0.016, 0.017 or 0.064°2θ/sec.

Melting properties disclosed herein were obtained from DSC thermograms,recorded with a heat flux DSC822e instrument (Mettler-Toledo GmbH,Switzerland). The DSC822e was calibrated for temperature and enthalpywith a small piece of indium (m.p.=156.6° C.; □Hf=28.45 J·g⁻¹). Sampleswere sealed in standard 40 μL aluminum pans, pin-holed and heated in theDSC from 25° C. to 300° C., at a heating rate of 10° C. min⁻¹. Dry N₂gas, at a flow rate of 50 mL min⁻¹ was used to purge the DSC equipmentduring measurement.

Mass loss due to solvent or water loss from the various crystal samplesdisclosed herein was determined by TGA/SDTA. Monitoring the sampleweight, during heating in a TGA/SDTA851e instrument (Mettler-ToledoGmbH, Switzerland), resulted in a weight vs. temperature curve. TheTGA/SDTA851e was calibrated for temperature with indium and aluminum.Samples were weighed into 100 μL aluminum crucibles and sealed. Theseals were pin-holed and the crucibles heated in the TGA from 25 to 300°C. at a heating rate of 10° C. min⁻¹. Dry N₂ gas was used for purging.

The gases evolved from the TGA samples were analyzed by a quadrupolemass spectrometer Omnistar GSD 301 T2 (Pfeiffer Vacuum GmbH, Germany),which analyses masses in the range of 0-200 amu.

Digital images disclosed herein were automatically collected for all thewells of each well-plate, employing a Philips PCVC 840K CCD cameracontrolled by Avantium Photoslider software.

HPLC analysis disclosed herein was performed using an Agilent 1200SLHPLC system equipped with UV and MS detectors following the conditionspresented below:

HPLC Equipment: LC-MS Manufacturer: Agilent HPLC: Agilent 1200UV-detector: Agilent DAD MS-detector: Agilent 1100 API-ES MSD VL-typeColumn: Waters Sunfire C18 (100 × 4.6 mm; 3.5 μm). Column temp: 30° C.Mobile phase: Gradient mode Mobile phase A: 1000/1; H₂O/TFA (v/v) Mobilephase B: 1000/5/1; ACN/MeOH/TFA (v/v) Flow: 1.0 mL/min Gradient program:Time [min]: % A: % B: 0   98  2 5   98  2 9   86 14 22   73 27 30   5050 30.10 98  2 Posttime: 7 UV-Detector: DAD Range: 200-400 nmWavelength: 244 nm Slit Width: 4 nm Time: 0-30 min MS-Detector: MSDScan: positive Mass Range: 70-1000 amu Fragmentator: 70 Time: 0-30 minAutosampler: Temperature: Not controlled Injection mode: loop Injectionvolume: 5 μL Needle wash: 2/3; ACN/H₂O (v/v) Dilution solvent: 0.1% TFAwater/CAN

Compound integrity disclosed herein is expressed as a “peak-area %” foreach peak (other than the peak due to injection), which is calculated bydividing the area of each peak in the chromatogram (“peak-area”) by thetotal peak-area (“total-area”) and multiplying by 100%, as follows:

${{peak} - {{area}{\mspace{11mu}\;}\%}} = {\frac{{peak} - {area}}{{total} - {area}}*100\%}$

The peak-area percentage of the compound of interest may be employed asan indication of the purity of the component in the sample.

Mass spectrometry disclosed herein was performed using a Finniganion-trap Mass Spectrometer Model LTQ XL. Samples were infused through asyringe pump into an atmospheric pressure electrospray ionization (ESI)probe. Fragmentation of the ions was achieved using collisionalactivation, and mass spectral data were collected in full scan (MS1) andmultilevel MS modes (MS2 and MS3). The structures of the product ionswere deduced using established fragmentation rules and through use ofMass Frontier software (High Chem Ltd., Slovak Republic, version5.1.0.3).

I. Polymorphic Forms of Brigatinib

Through analyses disclosed herein, ten polymorphic forms of brigatinibwere identified. The ten new polymorphic forms are referred to herein asForm A, Form B, Form C, Form D, Form E, Form F, Form G, Form H, Form J,and Form K. In general, crystalline forms of brigatinib have physicalproperties (such as high stability, etc.) that are advantageous for thecommercial preparation of solid dosage forms as compared to amorphousbrigatinib. The distinction between crystalline brigatinib and amorphousbrigatinib can be readily seen with the same type of physical chemicaldata (e.g., DSC, XRPD, thermal analysis) that is used to distinguish theindividual crystalline forms of brigatinib disclosed herein.

Form A:

Form A was the predominant crystalline form identified in theexperiments disclosed herein. Form A can be obtained from the the finalsynthetic step in the synthesis of brigatinib shown in FIG. 1, forexample, by elevating the temperature of crystallization to 60° C. andadding a NaOH solution at a slow rate. Form A is anhydrous and nothygroscopic. Form A did not convert into other forms viasolvent-mediated or solid-solid transition or exposure to elevatedtemperature, elevated humidity, mechanical pressure, or grinding asdisclosed herein.

The chemical and crystal structures of Form A have been unambiguouslyestablished by a combination of nuclear magnetic resonance spectroscopy(NMR), mass spectrometry (MS), and X-ray powder diffraction (XRPD),single crystal X-ray crystallography with confirmatory data fromelemental analysis (EA) and Fourier transform infra-red (FT-IR)spectroscopy.

In some embodiments, the present disclosure relates to crystalline FormA of brigatinib. In some embodiments, the present disclosure relates tocrystalline Form A of brigatinib, wherein the crystalline Form A ofbrigatinib is substantially pure. In some embodiments, the crystallineForm A is anhydrous.

Samples of Form A were analyzed by X-ray powder diffraction (XRPD). Insome embodiments, the present disclosure relates to crystalline Form Ahaving an x-ray powder diffraction pattern substantially as shown inFIG. 2.

In some embodiments, the XRPD pattern of crystalline Form A has at leastone, at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine, at least ten,at least eleven, at least twelve, at least thirteen, at least fourteen,at least fifteen, at least sixteen, or at least seventeen peaksexpressed in degrees two-theta chosen from 6.1, 8.6, 9.6, 10.8, 11.3,13.5, 14.3, 15.9, 17.2, 18.9, 19.4, 20.1, 21.8, 22.6, 23.1, 23.9, and27.7. As previously noted, in some embodiments, a variance of ±0.3 °2θmay be observed in one or more 2-θ peak positions.

In some embodiments, the XRPD pattern of crystalline Form A has at leastone, at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine, at least ten,at least eleven, at least twelve, at least thirteen, at least fourteen,at least fifteen, at least sixteen, or at least seventeen peaksexpressed in degrees two-theta chosen from 6.1, 8.58, 9.58, 10.78,11.34, 13.46, 14.34, 15.9, 17.22, 18.86, 19.38, 20.1, 21.82, 22.58,23.14, 23.86, and 27.66. A variance of ±0.30 °2θ may be observed in oneor more 2-θ peak positions in some embodiments.

In some embodiments, the present disclosure relates to the crystallineForm A having an x-ray powder diffraction pattern with at least one, atleast two, at least three, at least four, at least five, or at least sixpeaks expressed in degrees two-theta chosen from 9.6, 17.2, 19.4, 20.1,23.1, and 27.7. In some embodiments, a variance of ±0.3 °2θ may beobserved in one or more 2-θ peak positions.

In some embodiments, the present disclosure relates to the crystallineForm A having an x-ray powder diffraction pattern with at least one, atleast two, at least three, at least four, at least five, or at least sixpeaks expressed in degrees two-theta chosen from 9.58, 17.22, 19.38,20.1, 23.14, and 27.66. In some embodiments, a variance of ±0.30 °2θ maybe observed in one or more 2-θ peak positions.

In a differential vapor sorption (DVS) experiment with Form A, thesample was first dried at 0% RH for 6 hours. Then, the relative humiditywas cycled from 5% to 95% RH (sorption), then to 5% RH (desorption) at aconstant temperature of 25° C., with a hold time of 60 minutes per step.As shown in FIG. 3, these results demonstrate that Form A is nothygroscopic.

With reference to FIG. 4, the melting point of Form A was determined bydifferential scanning calorimetry (DSC). A sample of Form A was analyzedin a pin-holed 40 μL aluminum pan in the temperature range of 25° C. to300° C. at a heating rate of 10° C./min. An endothermic peak at 214.5°C. was observed. Accordingly, in some embodiments, the presentdisclosure relates to crystalline Form A having an onset meltingtemperature of 214.5° C. In some embodiments, the onset meltingtemperature of crystalline Form A is 214° C. In some embodiments, theonset melting temperature of crystalline Form A is 215° C.

With reference to FIG. 5, thermogravimetric analysis/single differentialthermal analysis (TGA/SDTA) and thermogravimetric mass spectrometry(TGMS) were performed on Form A. The sample, contained in a pin-holedcrucible, was heated in the TGA instrument from 25° C. to 300° C. at aheating rate of 10° C. min⁻¹, with dry N₂ gas used for purging. Gasesevolved from the TGA were analyzed using a quadrupole mass spectrometer.The TGA/TGMS experiment indicated that a mass loss of 0.23% (water) wasobserved over a temperature range of 30° C.-100° Cy.

Elemental analysis was performed on a Form A sample for hydrogen,carbon, nitrogen, chlorine, phosphorous and oxygen. The results areshown in Table 1 and confirm the molecular formula of brigatinib asC₂₉H₄₀ClN₇O₂P. The determined elemental composition is consistent withthe molecular formula of brigatinib.

TABLE 1 Elemental Analysis Results Element Actual Theoretical hydrogen 7.01%  6.73% carbon 58.88% 59.63% nitrogen 16.73% 16.79% chlorine 5.86%  6.07% phosphorous  5.14%  5.30% oxygen  6.38%  5.48%

Solution phase NMR studies were performed on Form A to obtain a completeassignment of ¹H, ¹³C and ³¹P resonances, and hence to confirm thechemical formula of brigatinib. ¹H NMR analyses were performed on asample of Form A dissolved in CD₃OD solvent, while ¹³C NMR analyses wereperformed on a sample of Form A dissolved in CDCl₃ solvent. FIG. 6provides the 1D ¹H-NMR spectra of Form A. FIG. 73 shows the 1D ¹³C-NMRspectra of Form A.

Table 2 summarizes the relevant chemical shift data of Form A obtainedfrom the ¹H, and ¹³C-NMR experiments. The number of signals and theirrelative intensity (integrals) confirm the number of protons and carbonsin the structure of Form A of brigatinib. The ³¹P-NMR chemical shift forthe single phosphorous atom in brigatinib was 43.6 ppm. These ¹H and¹³C-NMR chemical shift data are reported according to the atom numberingscheme shown immediately below:

TABLE 2 ¹H and ¹³C Chemical Shift Data (in ppm) of Form A of Brigatinib¹H Atom ¹³C Atom ¹³C, Number ¹H, ppm Letter ppm 1 1H, 8.0  A 18-19 2 1H,6.65 B  28.1 3 3H, 3.8  C  61.6 4 3H, 2.3  D 46-56 5 6H, 1.8-1.9 E 157.76 2H, 3.66-3.70 F 154.8 7 1H, 8.3  G 155.8 8 1H, 6.5  H 101-149 9 1H,7.2  — — 10 3H, 7.5-7.7 — — 11 17H, 1.0-3.0, — — unassigned

With reference to FIG. 8, mass spectral experiments of Form A werecarried out using an Agilent electrospray time of flight massspectrometer (Model 6210) operating in positive ion mode using flowinjection sample introduction. Samples of Form A were dissolved inmethanol/water and were analyzed and the mass observed was m/z 584.263(M+H⁺) with the calculated exact mass being 584.2664 (M+H⁺). Theobserved molecular mass is consistent with the elemental compositioncalculated from the molecular formula of brigatinib.

Using the Finnigan ion-trap mass spectrometer described above,fragmentation of the ions was achieved using collisional activation, andmass spectral data were collected in full scan (MS1) and multilevel MSmodes (MS2 and MS3) as shown in FIG. 9. The structures of the productions were deduced using established fragmentation rules and through useof Mass Frontier software (High Chem Ltd., Slovak Republic, version5.1.0.3) as shown in Table 3. The proposed structures of the key productions were consistent with the structure of brigatinib as shown in Table4.

TABLE 3 Mass Spectral Product Ions of Brigatinib Ion Selected forCollisional Experiment Activation Key Product Ions (m/z) MS Full Scan584 (M + H⁺) molecular ion MS² 584 484, 456, 452 (MS/MS) MS³ 484 467,456, 452, 448, 430, (MS/MS/MS) 416, 315, 297, 219 MS³ 456 424, 420, 406,388, 379, (MS/MS/MS) 297, 262, 185, 160 MS³ 452 435, 416, 387, 340, 299(MS/MS/MS)

TABLE 4 Mass Spectral Data of Product Ions of Brigatinib amu m/z ofDifference Chemical the from Groups Lost Product Precursor fromPrecursor Ion Proposed Strucure of the Ion Ion Ion MSExperiment—Molecular Ion m/z 584 584

 0 Molecular ion MH⁺. Calculation based on the nominal monoisotopicmolecular weight with ³⁵Cl atom. MS² Experiment—Product Ions of theMolecular Ion m/z 584 484

100 N-methyl piperazine 456

128 N-methyl piperazine and ethylene 452

132 (100 + 32) N-methyl piperazine and CH₃OH MS³ Experiment—Product Ionsof the Precursor Ion m/z 484 467

 17 *OH 456 Identical to that produced in MS² experiment m/z 584 →  28C₂H₄ 452 Identical to that produced in MS² experiment m/z 584 →  32CH₃OH 448

 36 HCl 430

 54 C₄H₆ 416

 68 (32 + 36) CH₃OH, HCl 315

169 2-dimethyl phosphoryl aniline 297

187 C₁₂H₁₃NO 219

265 C₁₂H₁₃NO, (CH₃)₂PO* MS³ Experiment—Product Ions of the Precursor Ionm/z 456 424

 32 CH₃OH 420

 36 HCl 406

 50 *HNCl 388

 68 HCl, CH₃OH 379

 77 (CH₃)₂PO* 297

159 C₁₀H₉NO 262

194 C₁₀H₉NOCl 185

271 C₁₀H₉NO, HCl, (CH₃)(CH₂)PO 160

296 C₁₂H₁₄N₄OPCl MS³ Experiment—Product Ions of the Precursor Ion m/z452 435

 17 *OH 416

 36 HCl 387

 65 *Cl, CH₂O 340

112 HCl, (CH₂)₂PO* 299

153 2-dimethyl phosphoryl phenyl

Single-crystal X-ray diffraction was employed to solve the crystalstructure of Form A of brigatinib. Crystals of brigatinib Form A wereobtained from MeOH-toluene, the structure of Form A brigatinib is shownin FIG. 10, and crystallographic parameters are summarized in Table 5.The structure is composed of hydrogen-bonded dimers. Based on thisstructure solution, it was determined that Form A is unsolvated. Somedisorder in the crystal is associated with the terminal N-methylpiperidine moiety of brigatinib.

TABLE 5 Crystal Data and Structure Refine- ment for Brigatinib Form AEmpirical formula C₂₉H₃₉ClN₇O₂P FW 584.11 Space group P-1 (No. 2) Unitcell dimensions: a [Å]  9.5619 (11) b [Å] 10.8027 (13) c [Å] 14.9715(17) α [°] 75.685 (5) □ [°] 79.835 (6) γ [°] 74.187 (5) V [Å³] 1431.8(3) Z 2 D_(c) [g/cm³] 1.355 Crystal size [mm³] 0.20 × 0.20 × 0.02Temperature (K) 150 Radiation (wavelength, Å) Cu K_(α) (1.54184)Monochromator confocal optics Linear abs coef, mm − 1 2.035 Absorptioncorrection applied empirical Transmission factors: min, max 0.79, 0.96Diffractometer Rigaku RAPID-II h, k, I range −11 to 9  −12 to 12 −17 to17 2θ range, deg 13.49-133.23 Mosaicity, deg 0.93 Programs used SHELXTLF₀₀₀ 620.0 Weighting 1/[σ²(Fo²) + (0.0806P)² + 0.0000P] where P = (Fo² +2Fc²)/3 Data collected 20289 Unique Data 4179 R_(int) 0.079 Data used inrefinement 4179 Cutoff used in R-factor F_(o) ² > 2.0σ (F_(o) ²)calculations Data with I > 2.0σ (I) 2420 Refined extinction coef 0.0034Number of variables 419 Largest shift/esd in final cycle 0.00 R (F_(o))0.063 Rw (F_(o) ²) 0.139 Goodness of fit 1.010

The % transmittance FT-IR spectrum of brigatinib Form A is shown inTable 6, with a summary of selected IR band assignments provided inTable 6. Data was collected on a Form A sample within a potassiumbromide salt plate.

TABLE 6 Selected IR Band Assignment of Brigatinib Assignment Frequency(cm⁻¹) Stretches for benzene and aliphatic 3241.0, 3165.1 and aromaticamines Stretches for alkane bonds 2980.0 to 2793.2 1,2 and 1,2,4substituted benzene 1616.4 to 1417.6 Aromatic nitrogen 1441.1 to 1219.8Aromatic ester 1354.6 to 1278.0 Aromatic chlorine 1307.4 to 1196.1Phosphoryl group 1163.6 to 1135.0 Alkane stretches 1094.9 to 794.6  1,2and 1,2,4 substituted 867.4 benzene Aliphatic secondary amines 768.6 to716.8

In some embodiments, the present disclosure relates to crystalline FormA having an FT-IR spectrum with any at least one of the followingfrequency bands:

Frequency (cm⁻¹) 3241.0, 3165.1 2980.0 to 2793.2 1616.4 to 1417.6 1441.1to 1219.8 1354.6 to 1278.0 1307.4 to 1196.1 1163.6 to 1135.0 1094.9 to794.6  867.4 768.6 to 716.8Form B:

Form B is hygroscopic. Form B can be obtained, for example, indirectlyfrom dehydration of hydrated Forms C and D. A mixture of Forms A, B, andC can form through vapor diffusion onto solids using water as thesolvent. None of the direct crystallization experiments disclosed hereinafforded Form B.

Form B can convert to the hydrated Forms C and D depending, for example,on the humidity level (e.g., above 60% RH at 30° C.). That conversionwas determined to be reversible. Form B converts irreversibly viasolid-solid transition to Form A at about 150° C. at ambient humidity asevidenced by XRPD. Form B also transforms to Form A upon slurrying inaqueous media at high temperature, for example, at least 37° C. Thesolubility of Form B could not be determined via slurries as Form Bconverted either to Forms D and/or C (at 25° C.) or Form A (at 37° C.).

In the DSC thermogram shown in FIG. 11, a minor endotherm was observedup to approximately 50° C., corresponding to water loss of some smallquantity of Form C present in the sample. Thereafter, Form B transformedvia solid-solid transition (exotherm shown at 171.8° C.) to Form A,which then melted (endotherm shown at 214.3° C.). That series of eventswas confirmed by VT-XRPD experiments on Form B.

Two cyclic DSC experiments using Form B were performed. In the firstexperiment, the temperature was elevated by 10° C./min to 190° C. andsubsequently decreased by 10° C./min to 25° C. as shown in FIG. 12. Theendotherm at around 70° C. in FIG. 12 can be attributed to the presenceof a small quantity of Form C and its water loss. The exotherm at 161°C. can be attributed to the solid-solid transformation of Form B to FormA. XRPD analysis of the solids at the end of the cyclic DSC experimentsconfirmed that the solid had transformed to Form A.

The second cyclic DSC experiment was performed with the followingthermal profile: heating by 10° C./min to 190° C., cooling by 10° C./minto 25° C.; second heating by 10° C./min to 300° C. The obtainedthermogram is shown in FIG. 13. The top thermogram is plotted vs. timeand the bottom thermogram is plotted vs. temperature. For the firstheating and cooling segments, the behavior was as described above forthe first cyclic DSC experiment. Upon the second heating, only themelting of Form A was observed at T_(peak)=214.0° C.

In some embodiments, the present disclosure relates to crystalline FormB of brigatinib. In some embodiments, the present disclosure relates tocrystalline Form B of brigatinib, wherein the crystalline Form B ofbrigatinib is substantially pure.

Samples of Form B were analyzed by X-ray powder diffraction (XRPD). Insome embodiments, the present disclosure relates to crystalline Form Bhaving an x-ray powder diffraction pattern substantially as shown inFIG. 14.

In some embodiments, the XRPD pattern of crystalline Form B has at leastone, at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine, at least ten,at least eleven, at least twelve, or at least thirteen peaks expressedin degrees two-theta chosen from 5.7, 9.2, 11.5, 12.8, 14.5, 15.5, 16.9,17.7, 19.2, 20.4, 21.8, 23.2, and 29.5. In some embodiments, a varianceof ±0.3 °2θ may be observed in one or more 2-θ peak positions.

In some embodiments, the XRPD pattern of crystalline Form B has at leastone, at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine, at least ten,at least eleven, at least twelve, or at least thirteen peaks expressedin degrees two-theta chosen from 5.74, 9.22, 11.46, 12.82, 14.5, 15.46,16.94, 17.66, 19.22, 20.38, 21.78, 23.18, and 29.54. In someembodiments, a variance of ±0.30 °2θ may be observed in one or more 2-θpeak positions.

In some embodiments, the present disclosure is related to crystallineForm A having an x-ray powder diffraction pattern with at least twopeaks expressed in degrees two-theta chosen from 11.5, 14.5, 16.9, 19.2and 23.2. In some embodiments, a variance of ±0.3 °2θ may be observed inone or more 2-θ peak positions.

In some embodiments, the present disclosure is related to crystallineForm A having an x-ray powder diffraction pattern with at least twopeaks expressed in degrees two-theta chosen from 11.46, 14.5, 16.94,19.22 and 23.18. In certain embodiments, th In some embodiments, avariance of ±0.30 °2θ may be observed in one or more 2-θ peak positions.

Form C:

Form C can be obtained, for example, from either partial dehydration ofhepta-hydrated Form D or by hydration of Form B. Form C is a hydratethat dehydrates to Form B upon exposure to relative humidity levelsbelow 25% RH at 30° C. Form C converts to Form D upon exposure to 90% RHat 30° C. These conversions are reversible with hysteresis. Upontemperature increase at ambient humidity, Form C dehydrates to Form B,which converts irreversibly via solid-solid transition to Form A asmeasured by XRPD. No direct crystallization experiment as describedherein afforded Form C.

The DSC thermogram in FIG. 15 shows an endotherm that corresponds towater loss (as confirmed by TGMS) by which the solid form converted toForm B. Form B converted via solid-solid transition (exotherm at 159.6°C.) to Form A, which in turn melted (endotherm at 214.3° C.). Thatseries of events was confirmed by VT-XRPD experiments on Form C.

Two TGMS thermograms from different samples of Form C are shown in FIGS.16A/B and FIGS. 17A/B, each containing a TGA/SDTA plot on top and TGMSplot at bottom. These thermograms show water mass losses of 4.25% and6.14% respectively. The corresponding numbers of water molecules are1.44 and 2.12, suggesting a degree of hydration of 2.

Form C can be obtained as a mixture of Forms A, B, and C through vapordiffusion onto solids using water as solvent. A mixture of Forms A and Ccan be obtained by cooling crystallization with hot filtration using assolvent systems any one of acetone/water (50/50), water/methanol(50/50), and water/1,4-dioxane (50/50). Another route to formation ofForm C is evaporation from acetone/water (50/50) solvent.

In some embodiments, the present disclosure relates to crystalline FormC of brigatinib. In some embodiments, the present disclosure relates tocrystalline Form C of brigatinib, wherein the crystalline Form C ofbrigatinib is substantially pure.

Samples of Form C were analyzed by X-ray powder diffraction (XRPD). Insome embodiments, the present disclosure relates to crystalline Form Chaving an x-ray powder diffraction pattern substantially as shown inFIG. 18.

In some embodiments, the XRPD pattern of crystalline Form C has at leastone, at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine, at least ten,at least eleven, at least twelve, at least thirteen, at least fourteen,at least fifteen, at least sixteen peaks expressed in degrees two-thetachosen from 2.1, 2.5, 5.4, 9.9, 10.9, 12.9, 14.9, 15.9, 16.6, 17.3,17.9, 19.2, 20.6, 23.9, 26.8, and 27.4. As previously noted, in someembodiments, a variance of ±0.3 °2θ may be observed in one or more 2-θpeak positions.

In some embodiments, the XRPD pattern of crystalline Form C has at leastone, at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine, at least ten,at least eleven, at least twelve, at least thirteen, at least fourteen,at least fifteen, at least sixteen peaks expressed in degrees two-thetachosen from

2.1, 2.54, 5.42, 9.9, 10.9, 12.86, 14.86, 15.94, 16.62, 17.26, 17.9,19.18, 20.58, 23.94, 26.82, and 27.42. In some embodiments, a varianceof ±0.30 °2θ may be observed in one or more 2-θ peak positions.

In some embodiments, the XRPD pattern of crystalline Form C has at leastone, at least two, at least three, at least four, at least five, atleast six peaks expressed in degrees two-theta chosen from 5.4, 14.9,15.9, 17.3, 19.2, and 23.9. In some embodiments, a variance of ±0.3 °2θmay be observed in one or more 2-θ peak positions.

Form D:

Form D is a heptahydrate that can be obtained directly fromcrystallization with methonal as the solvent and water as theanti-solvent. Form D can also be obtained from Form B, via Form C, uponslurries in aqueous media and exposure to high relative humidity (90% orhigher, at 30° C.). Form D dehydrates (partially) to Form C at about 80%RH at 30° C. Upon temperature increase at ambient humidity, Form Ddehydrates to Form C as measured by XRPD.

In some embodiments, the present disclosure relates to crystalline FormD of brigatinib. In some embodiments, the present disclosure relates tocrystalline Form D of brigatinib, wherein the crystalline Form D ofbrigatinib is substantially pure.

Samples of Form D were analyzed by X-ray powder diffraction (XRPD). Insome embodiments, the present disclosure relates to crystalline Form Dhaving an x-ray powder diffraction pattern substantially as shown inFIG. 19.

In some embodiments, the XRPD pattern of crystalline Form D has at leastone, at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine peaks expressedin degrees two-theta chosen from 4.7, 9.2, 9.7, 11.1, 14.5, 17.4, 18.9,22.4, and 23.7. As previously noted, in some embodiments, a variance of±0.3 °2θ may be observed in one or more 2-θ peak positions.

In some embodiments, the XRPD pattern of crystalline Form D has at leastone, at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine peaks expressedin degrees two-theta chosen from 4.66, 9.22, 9.74, 11.06, 14.54, 17.38,18.94, 22.42, and 23.66. As previously noted, in some embodiments, avariance of ±0.30 °2θ may be observed in one or more 2-θ peak positions.

In some embodiments, the XRPD pattern of crystalline Form D has at leastone, at least two, at least three, at least four, at least five peaksexpressed in degrees two-theta chosen from 9.7, 11.1, 17.4, 18.9, and23.7. As previously noted, in some embodiments, a variance of ±0.3 °2θmay be observed in one or more 2-θ peak positions.

In some embodiments, the XRPD pattern of crystalline Form D has at leastone, at least two, at least three, at least four, at least five peaksexpressed in degrees two-theta chosen from 9.74, 11.06, 17.38, 18.94,and 23.66. In some embodiments, a variance of ±0.3 °2θ may be observedin one or more 2-θ peak positions.

Conversion of Forms A-D:

Once Form A is obtained, no conventional method disclosed herein wasfound to convert this form to another form. Forms B, C and D, however,all interconverted depending on the temperature and relative humidityconditions.

At 30° C., increasing the humidity lead to hydration of Form B to Form Cand eventually to Form D. The changes were reversible upon humiditydecrease and occurred with a hysteresis: Form B converted to Form C atabout 65% RH while Form C dehydrated to Form B at 25% RH. Similarly,Form C converted to Form D at about 90% RH while Form D partiallydehydrated to Form C at 80% RH.

At ambient humidity, increasing the temperature lead to dehydration ofForms C and D to the anhydrous Form B (at about 40° C.) and to Form Avia solid-solid transition at about 150° C. These conversions were notreversible: Form A remained stable upon temperature decrease.

Thermal stability and stability under moisture were assessed followingstorage for a maximum of 5 weeks at 50° C., 75° C. (for Form A) and 40°C./75% relative humidity (for both Forms A and B). Within this periodsamples were analyzed by XRPD and HPLC as follows: after 1 day, 3 days,1 week, 2 weeks, 3 weeks, 4 weeks and 5 weeks. Form A was physically andchemically stable under all tested conditions. Form B, however,converted to the hydrated Form C after 1 day in the climate chamber andsubsequently to Form A (partially) (data up to 3 weeks).

Form E:

Form E can be obtained from freeze-drying from chloroform, and is achloroform solvate. Form E can also be obtained as a mixture with Form Aby slurrying with chloroform. After several weeks at ambienttemperature, Form E may revert to Form A as measured by XRPD. Analysisby TGA/SDTA (FIG. 20A) indicated a mass loss of 23.4% in the temperaturerange of 40-120° C., corresponding to 1.5 chloroform molecules perbrigatinib molecule. According to the SDTA signal and the indicatedmelting point, the solid occurring upon desolvation is Form A.

In some embodiments, the present disclosure relates to crystalline FormE of brigatinib. In some embodiments, the present disclosure relates tocrystalline Form E of brigatinib, wherein the crystalline Form E ofbrigatinib is substantially pure.

Samples of Form E were analyzed by X-ray powder diffraction (XRPD). Insome embodiments, the present disclosure relates to crystalline Form Ehaving an x-ray powder diffraction pattern substantially as shown inFIG. 21.

In some embodiments, the XRPD pattern of crystalline Form E has at leastone, at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine, at least ten,at least eleven, at least twelve, at least thirteen, at least fourteen,at least fifteen, at least sixteen, at least seventeen, at leasteighteen, at least nineteen peaks expressed in degrees two-theta chosenfrom 9.1, 10.2, 11.2, 12.0, 13.7, 14.4, 15.8, 16.5, 17.4, 18.3, 19.2,21.6, 22.3, 23.1, 23.9, 26.0, 26.4, 25.8, and 29.3. In some embodiments,a variance of ±0.3 °2θ may be observed in one or more 2-θ peakpositions.

In some embodiments, the XRPD pattern of crystalline Form E has at leastone, at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine, at least ten,at least eleven, at least twelve, at least thirteen, at least fourteen,at least fifteen, at least sixteen, at least seventeen, at leasteighteen, at least nineteen peaks expressed in degrees two-theta chosenfrom 9.06, 10.22, 11.18, 11.98, 13.66, 14.42, 15.82, 16.54, 17.42,18.34, 19.22, 21.62, 22.3, 23.14, 23.9, 26.02, 26.42, 25.78, and 29.34.In some embodiments, a variance of ±0.30 °2θ may be observed in one ormore 2-θ peak positions.

In some embodiments, the XRPD pattern of crystalline Form E has at leastone, at least two, at least three, at least four, at least five peaksexpressed in degrees two-theta chosen from 9.1, 10.2, 15.8, 19.2, and23.9. In some embodiments, a variance of ±0.3 °2θ may be observed in oneor more 2-θ peak positions.

In some embodiments, the XRPD pattern of crystalline Form E has at leastone, at least two, at least three, at least four, at least five peaksexpressed in degrees two-theta chosen from 9.06, 10.22, 15.82, 19.22,and 23.9. In some embodiments, a variance of ±0.30 °2θ may be observedin one or more 2-θ peak positions.

Form F:

Form F was obtained from a freeze-drying experiment using TFE/water, andis a TFE solvate. Form F desolvated to give Form A upon heating orstorage at ambient conditions for 8 weeks as measured by XRPD. Analysisby TGA/SDTA (FIG. 22) indicated a mass loss of 17.5% in the temperaturerange of 40-160° C., corresponding to 1.24 trifluoroethanol moleculesper brigatinib molecule. According to the SDTA signal and the indicatedmelting point, the solid occurring upon desolvation is Form A.

In some embodiments, the present disclosure relates to crystalline FormF of brigatinib. In some embodiments, the present disclosure relates tocrystalline Form F of brigatinib, wherein the crystalline Form F ofbrigatinib is substantially pure.

Samples of Form F were analyzed by X-ray powder diffraction (XRPD). Insome embodiments, the present disclosure relates to crystalline Form Fhaving an x-ray powder diffraction pattern substantially as shown inFIG. 22.

In some embodiments, the XRPD pattern of crystalline Form F has at leastone, at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine, at least ten,at least eleven, at least twelve, at least thirteen peaks expressed indegrees two-theta chosen from 8.5, 9.8, 11.1, 16.3, 17.0, 17.6, 18.7,19.4, 20.3, 22.0, 23.2, 23.9, and 27.1. In some embodiments, a varianceof ±0.3 °2θ may be observed in one or more 2-θ peak positions.

In some embodiments, the XRPD pattern of crystalline Form F has at leastone, at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine, at least ten,at least eleven, at least twelve, at least thirteen peaks expressed indegrees two-theta chosen from 8.46, 9.78, 11.14, 16.34, 17.02, 17.58,18.74, 19.38, 20.34, 22.02, 23.22, 23.86, and 27.1. In some embodiments,a variance of ±0.30 °2θ may be observed in one or more 2-θ peakpositions.

In some embodiments, the XRPD pattern of crystalline Form F has at leastone, at least two, at least three, at least four, at least five peaksexpressed in degrees two-theta chosen from 9.8, 17.0, 19.4, 20.3, and27.1. In some embodiments, a variance of ±0.3 °2θ may be observed in oneor more 2-θ peak positions.

In some embodiments, the XRPD pattern of crystalline Form F has at leastone, at least two, at least three, at least four, at least five peaksexpressed in degrees two-theta chosen from 9.78, 17.02, 19.38, 20.34 and27.1. In some embodiments, a variance of ±0.30 °2θ may be observed inone or more 2-θ peak positions.

Form G:

Form G was obtained from a crash crystallization experiment, withchloroform as solvent and acetonitrile as anti-solvent. Form G inmixture with Form A was also obtained from two other experiments usingchloroform (anti-solvent addition and thermocycling). Remeasurement byXRPD of Form G, after storage of the measuring plate at ambientconditions for 5 weeks, showed that Form G had transformed to Form A.Form G may be an instable form, and may, for example, be a chloroformsolvate, which desolvates and converts to Form A upon storage at ambientconditions.

In some embodiments, the present disclosure relates to crystalline FormG of brigatinib. In some embodiments, the present disclosure relates tocrystalline Form G of brigatinib, wherein the crystalline Form G ofbrigatinib is substantially pure.

Samples of Form G were analyzed by X-ray powder diffraction (XRPD). Insome embodiments, the present disclosure relates to crystalline Form Ghaving an x-ray powder diffraction pattern substantially as shown inFIG. 24.

In some embodiments, the XRPD pattern of crystalline Form G has at leastone, at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine, at least ten,at least eleven, at least twelve, at least thirteen, at least fourteenpeaks expressed in degrees two-theta chosen from 7.2, 8.3, 9.7, 10.4,12.9, 15.8, 18.1, 18.7, 20.7, 21.5, 22.8, 23.5, 24.5, and 26.8._In someembodiments, a variance of ±0.3 °2θ may be observed in one or more 2-θpeak positions.

In some embodiments, the XRPD pattern of crystalline Form G has at leastone, at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine, at least ten,at least eleven, at least twelve, at least thirteen, at least fourteenpeaks expressed in degrees two-theta chosen from 7.22, 8.34, 9.7, 10.38,12.86, 15.78, 18.1, 18.7, 20.74, 21.46, 22.82, 23.54, 24.5, and 26.82.In some embodiments, a variance of ±0.30 °2θ may be observed in one ormore 2-θ peak positions.

In some embodiments, the XRPD pattern of crystalline Form G has at leastone, at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight peaks expressed in degreestwo-theta chosen from 8.3, 9.7, 12.9, 15.8, 18.1, 20.7, 22.8, and 26.8.In some embodiments, a variance of ±0.3 °2θ may be observed in one ormore 2-θ peak positions.

In some embodiments, the XRPD pattern of crystalline Form G has at leastone, at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight peaks expressed in degreestwo-theta chosen from_8.34, 9.7, 12.86, 15.78, 18.1, 20.74, 22.82 and26.82. In some embodiments, a variance of ±0.30 °2θ may be observed inone or more 2-θ peak positions.

Form H:

Form H can be obtained as a pure form or as a mixture with Form Athrough a cooling-evaporative method from a variety of solvents, such asfor example ethanol/water, 1,4 dioxane/water, methanol,methanol/chloroform, and methanol/acetonitrile. Form H may be a solvatethat accommodates small alcohols such as methanol, ethanol, and1,4-dioxane. After storage at ambient conditions for 1-3 weeks, Form Hhad partially transformed to Form A as determined by XRPD.

In some embodiments, the present disclosure relates to crystalline FormH of brigatinib. In some embodiments, the present disclosure relates tocrystalline Form H of brigatinib, wherein the crystalline Form H ofbrigatinib is substantially pure.

Samples of Form H were analyzed by X-ray powder diffraction (XRPD). Insome embodiments, the present disclosure relates to crystalline Form Hhaving an x-ray powder diffraction pattern substantially as shown inFIG. 25.

In some embodiments, the XRPD pattern of crystalline Form H has at leastone, at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine, at least ten,at least eleven, at least twelve peaks expressed in degrees two-thetachosen from_4.2, 5.2, 8.4, 10.9, 12.7, 15.0, 15.7, 16.5, 17.2, 18.4,19.5, and 21.3. In some embodiments, a variance of ±0.3 °2θ may beobserved in one or more 2-θ peak positions.

In some embodiments, the XRPD pattern of crystalline Form H has at leastone, at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine, at least ten,at least eleven, at least twelve peaks expressed in degrees two-thetachosen from 4.22, 5.22, 8.38, 10.86, 12.66, 14.98, 15.74, 16.5, 17.18,18.42, 19.5, and 21.3. In some embodiments, a variance of ±0.30 °2θ maybe observed in one or more 2-θ peak positions.

In some embodiments, the XRPD pattern of crystalline Form H has at leastone, at least two, at least three, at least four, at least five, atleast six peaks expressed in degrees two-theta chosen from 4.2, 5.2,8.4, 10.9, 12.7, and 21.3._In some embodiments, a variance of ±0.3 °2θmay be observed in one or more 2-θ peak positions.

In some embodiments, the XRPD pattern of crystalline Form H has at leastone, at least two, at least three, at least four, at least five, atleast six peaks expressed in degrees two-theta chosen from 4.22, 5.22,8.38, 10.86, 12.66, and 21.30. In some embodiments, a variance of ±0.30°2θ may be observed in one or more 2-θ peak positions.

Form J:

Form J was obtained as a mixture with Form A from 2-methoxyethanol in acooling evaporative experiment at μL scale. Remeasurement by XRPD of themixture of Forms A+J, after storage of the measuring plate at ambientconditions for 3 weeks, showed that the material was still a mixture ofForms A+J; however, the component of Form A was clearly larger.

The mixture of Form A and Form J was analyzed by X-ray powderdiffraction (XRPD) and the pattern is shown in FIG. 26. The XRPD patternhas at least one or all of the following peaks in degrees two theta (2θ)is shown for Forms A+J: 5.3, 7.6, 11.2, 17.6, 18.5, 19.8, and 21.3. Incertain embodiments, the mixture of Forms A+J is characterized by a XRPDpattern comprising one or more of the following peaks in degrees twotheta (2θ): 7.6, 17.6, and 21.3. In certain embodiments, the XRPDpattern of the mixture of Forms A+J can have two peaks or three peaks ofthe above-listed peaks.

Forms K and L:

Forms K and L were obtained as mixtures with Form A and their XRPDpatterns exhibit only minor differences with that of Form A. Form K wasobtained as a mixture with Form A from THF/NMP mixture in a coolingevaporative experiment at μL scale. Remeasurement by XRPD of the mixtureof Forms A+K, after storage of the measuring plate at ambient conditionsfor 3 weeks, showed that the material was still a mixture of Forms A+K.

Form L was also obtained as a mixture with Form A from slurryexperiments with n-heptane, hexane or methylcyclohexane. Remeasurementby XRPD of the mixtures A+L, after storage of the measuring plate atambient conditions for 3 weeks, showed that the solids were still amixture of A+L.

FIGS. 27A and 27B show the XRPD patterns observed for the mixtures A+Kand A+L. The markers indicate the 2θ positions where the additionalintensity peaks appear. For Form K, the peaks that are additional toForm A, as described above, include in degrees two theta (2θ): 5.5, 7.7,and 12.3. For Form L, the peak that is additional to Form A, asdescribed above, in degrees two theta (2θ): 18.2. In certainembodiments, the XRPD patterns of either Form K or Form L can show twopeaks or three peaks of the above-listed peaks.

Amorphous Form of Brigatinib

Grinding experiments were performed to obtain amorphous brigatinib.After grinding a sample of Form A for 30 and 60 minutes, XRPD studiesindicated an increase in amorphous content as shown in FIG. 28. Puritywas assessed by HPLC and confirmed that chemical degradation did notoccur during the grinding process. In a mechanical stress test viagrinding, a sample of Form A was ground for 2, 3, 4 and 5 hours.Recovered solids were analyzed by XRPD and HPLC. By 5 hours, the samplewas almost completely amorphous.

II. Experiments Identifying Brigatinib Polymorphic Forms

Initial efforts to identify polymorphic forms of brigatinib were dividedinto two phases. Phase 1 included starting-material characterization,feasibility testing, solubility studies, compression studies, andintrinsic dissolution rate to provide data for the solvent selection forPhase 2. Phase 2 included polymorph screening experiments at milliliter(mL) and microliter (μL) scales. These efforts led to the identificationof 10 polymorphic forms: Form A, Form B, Form C, Form D, Form E, Form F,Form G, Form H, Form J, and Form K.

Phase 1: Starting Material Characterization

The starting material, brigatinib, was provided as an off-white solidand its chemical purity was assessed by HPLC as 99.9%. Mass spectraldata confirmed the molecular weight of brigatinib to be 584 g/mol. TGAand TGMS analyses showed 0.23% of mass loss (corresponding to about 0.08water molecules per Form A molecule) in the temperature interval of 30°C.-100° C. DSC analysis showed an endothermic event with T_(peak)=214.5°C., related to melting of the compound, brigatinib. The water content ofForm A was determined by coulometric Karl Fischer method. The averagewater content from two determinations was found to be 0.32%.Representative residual heavy metals in brigatinib Form A weredetermined by ICP-MS. The detected elements included cadmium (0.02 ppm),copper (0.14 ppm), molybdenum (0.10 ppm), palladium (0.087 ppm) andsilver (0.03 ppm). The following metals were not detected: antimony,arsenic, bismuth, lead, mercury and tin.

NaOH titration experiments were performed to investigate the influenceof the NaOH addition rate and crystallization temperature on theisolated crystal form. A stock solution of brigatinib Form A wasprepared by weighing in 450 mg of Form A and slurrying in 9 mL water for10 min. A quantity of 4.5 mL of 1M HCl was added to dissolve thebrigatinib (final API concentration 33.3 mg/mL). For each experiment, 3mL of stock solution was added in an 8 mL vial, containing a stirringbar, pH probe and tubing connected to the titrator (Titrino). The vialwas placed in the Crystalline and brought to temperature beforeinitiating NaOH titration. A volume of 3 mL 0.1M NaOH solution wastitrated at a predefined rate. During the experiment, bottom stirring at500 rpm was applied. While brown solids appeared during titration; uponstirring (10 min) the color changed to pink. Subsequently, all solidswere separated from the solution by centrifugation, washed two timeswith 5 mL of water and then dried.

Four sets of NaOH addition rate (mL/min) and temperature ° C. conditionswere evaluated: 0.02 mL/min at 25° C., 20 mL/min at 60° C., 0.05 mL/minat 25° C., and 20 mL/min at 60° C. Direct formation of Form A ispossible from aqueous media when the process occurs at 60° C. and a slowNaOH addition is applied. A fast NaOH addition led to a mixture of FormA and the hepta-hydrated Form D while at 25° C., the crystallized fromwas the heptahydrate independently of the NaOH addition rate.

Phase 1: Solubility Study

Quantitative solubility testing was performed on brigatinib startingmaterial, employing a set of 24 solvents (DMSO, heptane and water wereperformed in triplicate). In a vial, about 40 mg of starting material,400 μL of solvent and a stir bar were added. After stirring for 24 h at20° C. for 24 hours, the liquid was retrieved, filtered, and analyzedfor API content by HPLC. The residual solids were characterized by XRPDand found to be Form A. The results are summarized in Table 7.

TABLE 7 Solubility of Brigatinib Solubility XRPD Solvent name (mg/mL)Form¹ Acetone 0.69 Form A Acetonitrile 0.36 Form A 1-Butanol 17.74 FormA 2-Butanone 1.11 Form A Butyl acetate 0.32 Form A Chloroform¹ >181.8 —Cyclohexane UR², <0.01 Form A 1,2-Dichloroethane 38.29 Form ADichloromethane¹ >196.87 — 1,2-Dimethoxyethane 1.13 Form A DimethylSulfoxide 2.95 Form A Dimethyl Sulfoxide 3.02 Form A Dimethyl Sulfoxide3.05 Form A N,N-Dimethylacetamide 0.47 Form A 1,4-Dioxane 4.01 Form AEthanol 6.71 Form A Ethyl Acetate 0.42 Form A Ethyl Formate 0.99 Form An-Heptane UR, <0.01 Form A n-Heptane UR, <0.01 Form A n-Heptane UR,<0.01 Form A Isopropyl acetate UR, <0.01 Form A Methanol 35.31 Form ANitromethane 0.41 Form A Isopropanol 1.55 Form A Tetrahydrofuran UR,<0.01 Form A Water 0.09 Form A Water 0.09 Form A Water 0.09 Form Ap-Xylene 0.35 Form A 2,2,2-trifluoroethanol³ >224 —2,2,2-trifluoroethanol/water (90:10)³ >172 —2,2,2-trifluoroethanol/water (80:20)³ >159 — ¹Samples were dissolvedafter 24 h equilibration time, no solids were harvested. ²Under Range,lower then detection limit, the concentration is lower than 0.22 mg/mL³Data obtained from freeze drying experiment

The solubility of Form A was also evaluated in Simulated Gastric Fluidand observed to be 52 mg/ml. At 37° C. in aqueous buffers, solubilitiesof Form A were observed to be 70 mg/mL (in pH 1.0), 26 mg/mL (in pH 4.5)and 6 mg/mL (in pH 6.5).

In a second solubility study, the solubility of Forms A and B weredetermined in triplicate at 25° C. and 37° C. in water, pH 1.0 buffer(0.1 N HCl), pH 4.5 acetate buffer, pH 6.5 phosphate buffer andsimulated gastric fluid SGF at 37° C. For each medium, a standard 1.8 mLscrew cap vial was charged with circa 40 mg of the starting material,400 μl of solvent and a magnetic stirring bar (in the cases ofchloroform and dichloromethane 200 μl of solvent were used). The vialswere subsequently closed and equilibrated at the correspondingtemperature for 24 h while stirring. The liquid part was retrieved witha syringe and filtered (0.5 micron filter); the isolated mother liquorswere diluted to two dilutions selected according to the calibrationcurve. Quantities of the API in the diluted solutions were determinedvia HPLC analysis (DAD). The calibration curve was obtained from twoindependently prepared stock solutions of compound brigatinib in 50%water/50% acetonitrile/0.1% TFA. Subsequently, the separated solids weremeasured wet by XRPD to confirm the solid form of which the solubilitywas measured.

In Table 8, the solid forms of the separated slurries are listed. Form Aremained stable in all media, while Form B converted to the hydratedForms D and/or C in the experiments at 25° C. and to Form A in theexperiments at 37° C. At the latter temperature and in water, Form Bconverted to the hydrates C and D and not to A as in the rest of themedia. The solubility of Form B could not be measured as it converted toother solid forms. The average solubility values, shown in the sametable, refer to the solid form to which the initially placed Form B wastransformed. Hence, it was not possible to measure the solubility ofForm B but rather that of Form C (and of C+D). The solubility values areplotted in FIG. 29. The solubility is greater in acidic media comparedto basic ones.

TABLE 8 Form Attained at Solubility Study Conclusion Initial Form Form AForm B Temp 25° C. 37° C. 25° C. 37° C. Medium Form Solub (mg/mL) FormSolub (mg/mL) Form Solub (mg/mL) Form Solub (mg/mL) water A 0.11 ± 0.0 A 0.1 ± 0.0 C 0.1 ± 0.0 C + D 0.3 ± 0.0 pH 1.0 A 60.4 ± 2.9  A 70.6 ±1.5  C 68.6 ± 2.0  A 70.7 ± 0.6  pH 4.5 A 24.4 ± 1.2  A 26.0 ± 0.0  C +D 24.8 ± 1.4  A 25.1 ± 0.3  pH 6.8 A 8.6 ± 1.4 A 6.2 ± 0.1 C + D 13.2 ±1.3  A 6.0 ± 0.1 SGF — — A 51.7 ± 0.6  — — A 51.3 ± 0.3 

In a third solubility study, Form A was measured in different buffersolutions as shown in Table 9.

TABLE 9 Solubility Measurements of Form A in Buffers Slurry pH BufferConc. (mg/mL) 1.7-2 HCl/KCl 177 2.4 Potassium hydrogen phthalate/HCl 3293.6 Potassium hydrogen phthalate/HCl 173 6.2 KH₂PO₄/NaOH 8 7.2KH₂PO₄/NaOH 11Phase 1: Feasibility Study

Feasibility tests were performed to attempt to obtain amorphous startingmaterial that could be employed in some crystallization techniques ofthe Phase 2 portion of the study. Two techniques were employed, i.e.grinding and freeze-drying. The results are presented below.

Grinding. Two grinding experiments were performed on samples of Form Awith two different durations (30 and 60 min) at a frequency of 30 Hz.Their amorphous content increased with time, but their purity was stableat about 100%. Mechanical stress via grinding experiments were alsoperformed, with grinding times of 2, 3, 4, and 5 hours. Similarly,amorphous content increased without degradation of chemical purity.

Freeze-drying. Six freeze-drying experiments were performed with samplesof Form A as described in Table 10. Samples 1, 2, and 4 remained mostlycrystalline, but samples 3 and 5 were amorphous and contained about15-16% residual solvent. Sample 6 was amorphous and contained about 7%residual solvent. Forms E and F were produced using this method.However, due to the variable form and solvation, freeze-drying was notfurther employed to obtain amorphous brigatinib.

TABLE 10 Freeze-drying Feasibility Study of Brigatinib, Form A Solventvolume starting (μL) Con- Form Solvent material centration Obtainedcontent sample Solvent (mg) (mg/mL) (XRPD) (%) 1 Chloroform 19.9 100Form E 23.4 199 2 Dichloro- 19.9 100 Form A 2.23 methane 199 32,2,2-Trifluoro- 22.4 100 Form A + 15.0 ethanol 224 amorphous 4 2,2,2-17.2 100 Form F 17.5 Trifluoroethanol/ 172 Water 90/10 5 2,2,2- 15.9 100Form A + 16.1 Trifluoroethanol/ 159 amorphous Water 80/20 6 2,2,2- 20.2500 amorphous 6.9 Trifluoroethanol/ 40.4 Water 50/50Phase 1: Compression

Compression tests were performed on brigatinib Form A in order todetermine whether pressure-induced phase transformations or loss ofcrystallinity occurred. The press used was an Atlas Manual 25 TonHydraulic Press (from SPECAC). Experiments were carried out at 3 and 6ton/cm² for one minute in each case. The pressed solids were measured byXRPD and no phase transitions or peak shifts in the XRPD patterns wererevealed. The purity by HPLC of the two samples subjected to thecompression tests were both determined to be comparable to that of thestarting material.

Phase 1: Intrinsic Dissolution Rate

For measuring the intrinsic dissolution rate (IDR), the startingmaterial was tableted using a mini-IDR compression system (pION/HeathScientific). For preparation of the tablets, approximately 11 mg ofmaterial was pressed in the cylindrical hole of a passivated stainlesssteel die, to a uniform, flat surface, with an exposed area of 0.072cm². The pressure applied was approximately 50 bar for 3-5 min. Thesample die was inserted in a cylindrical Teflon rotating disk carriercontaining an embedded magnetic stirring bar at its base. Thedie/stirrer assembly was placed in a flat bottomed glass vial, ready fordissolution analysis.

The dissolution rate was measured in 20 mL of solvent (medium) and thepath length of the UV meter was 2 mm. Applied stirring speed duringmeasurement was 100 rpm. Measurements were performed at 20° C. and 37°C.

For determining the dissolution rate from a powdered sample,approximately 5 mg of brigatinib (Form A or B) was weighed into a 5 mLdissolution vial and the dissolution probe was inserted into the vial.Subsequently, 4 mL of water was added at the same time the measurementwas initiated. The concentration was recorded for 20 h.

In the first series, the IDR of Forms A and B were determined inmonoplicate. The measurements were carried out in at 25° C. and 37° C.in water, pH 1.0 (0.1 N HCl) buffer, pH 6.8 phosphate buffer and insimulated gastric fluid SGF. In FIGS. 30-37, the IDRs are plotted forcomparison between the forms and the same medium or between variousmedia and the same form. The IDR of each of Forms A and B in the variousmedia increases with increasingly acidic media (see FIGS. 34-37).

The intrinsic dissolution rate measurements of Form A in pH 1.0 and SGFshows that, within 5 min, roughly a concentration of 0.25 mg/ml could bereached. That indicates that, in the stomach, together with a 200 mlglass of water, about 50 mg of Form A could be dissolved (numbers areonly indicative).

Furthermore, the IDR experiments show that Form A remains stable whenslurried in water, SGF, and pH 1.0 aqueous buffer. Based on thoseresults, no conversion would be expected to take place in the stomach.

In several cases, the results were counter-intuitive. These results wererelated to (1) the dissolution rate of the compound at 25° C. beinghigher compared to that at 37° C. (in the cases of Form A in water andpH 6.5 buffer—for the first 3-4 min—and of Form B in pH 6.5 buffer—inthe whole range), while it is expected that the IDR at 37° C. would bethe highest; and (2) the IDR of Form A being higher compared to that ofForm B, at pH 6.5 buffer, while one would expect the opposite on thebasis of the relative stability of Forms A and B. To further study theseresults, two additional series of experiments were performed: (a) theIDR's of Forms A and B were measured (in monoplicate) in water at 25° C.and in pH 6.5 buffer at 25° C. and 37° C.; and (b) the IDR's of Form Awere measured in triplicate in water and pH 6.5 buffer at 25° C. Theresults of these additional experiments are plotted in FIGS. 38-42.

With respect to the first observation (IDR of Form A higher at 25° C.compared to that at 37° C.), the following comments can be made:

FIG. 30: The IDR of Form A in water at 25° C. appears to be higher forthe first 3 min. One possibility for this result is detachment of atablet grain, which adds to the concentration. Thereafter, theconcentrations of both Forms A and B are higher at 37° C., which is asexpected. However, remeasurements of the IDR of Form A in water at bothtemperatures, showed considerable variability (FIGS. 38A/B). Onepossibility for this result is the low concentrations, which make themeasurement more sensitive to measuring conditions.

FIG. 31: Similarly, the IDRs in pH 1.0 buffer of both Forms A and B at25° C. appear to be higher, one possibility is the detachment of tabletgrains, as the large increase in concentration (at about 1 min) in bothcases indicates. Concentrations higher than 0.25 mg/mL are not plottedas the detector reaches saturation at about these values.

FIGS. 32A/B: The IDR of Form A in pH 6.5 buffer at 25° C. appears to behigher than that at 37° C. for the first 4 min, one possibility is thedetachment of tablet grains; after 4 min, the IDR at 37° C. becomeshigher. However, remeasurements of the IDR of Form A in pH 6.5 buffer,showed that the rate was higher at 37° C. compared to that at 25° C.(FIGS. 39A/B). The IDR of Form B appears to be higher at 25° C.,however, the concentration of Form B at both temperatures appears to bestable. On a repetition of the IDR measurements of Form B in pH 6.5buffer, in the second series of experiments, the results showed that theIDR at 37° C. was higher than that at 25° C. (FIGS. 40A/B). However, itis possible there was variability in the measurements, again likely dueto the low concentrations, which make the measurement more sensitive tomeasuring conditions.

The observation that Form A appeared to dissolve faster than Form B(FIGS. 32A/B) was investigated in a second series of IDR measurements.In FIG. 41, all IDR measurements of Forms A and B at 25° C. are plotted:the second series of experiments showed that after 3 min, theconcentration of Form B is the highest, which is expected. Prior to 3min, large increases of concentration are observed in several cases,indicating grain detachments from the tablets. In FIGS. 42A/B, all IDRmeasurements of Forms A and B at 37° C. are plotted: the second seriesof experiments showed that after about 1 min, the concentration of FormB was the highest.

It is noted that, in the cases of the IDR's of Forms A and B in waterand pH 6.5 buffer, at both 25 and 37° C., the concentration values arevery low, making the recorded values very sensitive to the measuringconditions. Measurements at these concentrations are prone tovariability to a larger extent compared to measurements at higherconcentrations. These values of IDR should be taken as indicative ratherthan absolute.

FIGS. 33 A/B: The IDR of Form A in SGF at 25° C. appears to be higherthan that at 37° C. for the first 5 min, possibly from detachment of atablet grain in the beginning of the measurement which adds to theconcentration. Thereafter, the IDR's of Form A at both 25 and 37° C.appear similar. The IDR of Form B is higher at 37° C. than at 25° C., asexpected. In pH 1.0 buffer and SGF, the IDRs of both Forms A and B andat both temperatures are comparable (see FIG. 31 and FIGS. 33A/B). Atconcentrations around 0.3 mg/mL, the detector is close to saturation.

FIG. 34: Plots of increasing concentration of Form A vs. time from IDRexperiments at 25° C. in water and aqueous buffers of pH 1.0, 4.5 and6.5.

FIG. 35: Plots of increasing concentration of Form A vs. time from IDRexperiments at 37° C. in water and aqueous buffers of pH 1.0, 4.5 and6.5.

FIG. 36: Plots of increasing concentration of Form B vs. time from IDRexperiments at 25° C. in water and aqueous buffers of pH 1.0, 4.5 and6.5.

FIG. 37: Plots of increasing concentration of Form B vs. time from IDRexperiments at 37° C. in water and aqueous buffers of pH 1.0, 4.5 and6.5.

For measuring the dissolution rate from powder, the test was onlyperformed in water for Forms A and B at 37° C., as the solubilities ofForms A and B were low enough to permit detection.

In FIGS. 43 A/B, the concentration of Forms A and B vs. time areplotted. In both cases, within about 10 min, the concentration reached a“maximum” and thereafter the dissolution was slowed down. Between 10 minand 20 h, the concentration of Form A almost doubled (from 0.07 to 0.14mg/mL). For Form B, between 10 min and 260 min, a concentration decreasewas observed; thereafter, the concentration increased again, to reach atthe end of the experiment a value slightly higher compared to that at 10min. The concentration increase might be connected to the transformationof Form B to Form D, which re-dissolved. Due to detector saturation atabout 0.3 mg/mL, the maximum concentration of Form B was notconclusively determined.

Phase 2: Polymorph Identification

The polymorph screening experiments for brigatinib were carried out atmilliliter (mL) scale using nearly 300 different conditions and also atmicroliter scale using nearly 200 different conditions. Six differentcrystallization procedures were applied: (1) cooling-evaporation; (2)evaporative crystallization; (3) vapor exposure; (4) coolingcrystallization with hot filtration; (5) crash crystallization withanti-solvent addition; (6) slurry; (7) vapor diffusion into solution;(8) vapor diffusion onto solids; (9) grinding; (10) thermocycling; (11)VT-XRPD; (12) VH-XRPD; (13) DVS; and (14) dehydration. After thescreening experiments were completed, the materials were collected andanalyzed by XRPD and digital imaging.

Cooling-Evaporative Crystallization Experiments

The cooling-evaporative experiments shown at Tables 11-14 at μL scalewere performed in 96-well plates, employing 24 different solvents andsolvent mixtures, 2 concentrations, and 2 temperature profiles. In eachwell, 4 mg of Form A was weighed. Then, the screening solvent was addedto reach a concentration of circa 40 mg/mL or 80 mg/mL. The plates, witheach well individually sealed, were placed in a CrystalBreeder™ toundergo a temperature profile as described in Table 10 below. The plateswere then placed under vacuum and evaporated for several days under 200mbar and/or 5 mbar, then analyzed by XRPD and digital imaging. The finalForm obtained is given in Tables 12-14.

TABLE 11 Cooling-evaporative crystallization parameters CoolingT^(initial) Hold rate T^(final) Ageing Experiment T profile (° C.) (min)(° C./h) (° C.) (h)  1-48 T1 60 60 1 5 48  49-96, T2 60 60 20 5 3145-192  97-144 T3 60 60 1 20 48 193-240 T4 60 60 20 20 3

TABLE 12 Cooling-evaporative crystallization experimental results: T1and T2 profiles T profile 1 Tprofile 2 Conc (mg/mL) 40 80 40 80 SolventExpt. Form Expt. Form Expt. Form Expt. Form tert-Butyl methyl ether 1 A25 A 49 A 73 A Methyl acetate 2 A 26 A 50 A 74 A Methanol 3 A + H 27 A +H 51 A + H 75 A Tetrahydrofuran 4 A 28 A 52 A 76 A Acetonitrile 5 A 29 A53 A 77 A 1,2-Dimethoxyethane 6 A 30 A 54 A 78 A Isopropyl acetate 7 A31 A 55 A 79 A 1,4-Dioxane 8 A 32 A 56 A 80 A 2-Methoxyethanol 9 A + J33 A 57 A 81 A → A + J 2-Hexanone 10 A 34 A 58 A 82 A Heptane 11 A 35 A59 A 83 A 1-Pentanol 12 A 36 A 60 A 84 A Acetone/Dichloromethane (50/50)13 A 37 A 61 A 85 A Methanol/Chloroform (50/50) 14 A + H 38 A 62 A + H86 A tert-Butyl methyl ether/Chloroform (50/50) 15 A 39 A 63 A 87 AMethanol/Acetonitrile (50/50) 16 A 40 A 64 A 88 AAcetonitrile/Chloroform (50/50) 17 A 40 A 65 A 89 A Heptane/Ethylformate (50/50) 18 A 42 A 66 A 90 A 1,4-Dioxane/Cyclohexane (50/50) 19 A43 A 67 A 91 A Water/Methanol (50/50) 20 A 44 A 68 A 92 ACyclohexane/N-Methylpyrrolidone (50/50) 21 A 45 A 69 A 93 ATetrahydrofuran/N-Methylpyrrolidone (50/50) 22 A 46 A 70 A 94 A1,2,3,4-Tetrahydronaphthalene/Acetonitrile (50/50) 23 A 47 A 71 A 95 AChlorobenzene/N-Methylpyrrolidone (50/50) 24 A 48 A 72 A 96 A

TABLE 13 Cooling-evaporative crystallization experimental results: Tprofile 3 T profile 3 Conc. (mg/mL) 40 80 40 80 Solvent Expt. Form Expt.Form Expt. Form Expt. Form tert-Butyl methyl ether 97 A 121 A 145 A 169A Methyl acetate 98 A 122 A 146 A 170 A Methanol 99 A + H 123 A + H 147A + H 171 A + H Tetrahydrofuran 100 A 124 A 148 A 172 A Acetonitrile 101A 125 A 149 A 173 A 1,2-Dimethoxyethane 102 A 126 A 150 A 174 AIsopropyl acetate 103 A 127 A 151 A 175 A 1,4-Dioxane 104 A 128 A 152 A176 A 2-Methoxyethanol 105 A 128 A 153 A 177 A 2-Hexanone 106 A 130 A154 A 178 A Heptane 107 A 131 A 155 A 179 A 1-Pentanol 108 A 132 A 156 A180 A Acetone/Dichloromethane (50/50) 109 A 133 A 157 A 181 AMethanol/Chloroform (50/50) 110 H → 134 n 158 H → 182 H → A + H A + HA + H tert-Butyl methyl ether/Chloroform (50/50) 111 A 135 A 159 A 183 AMethanol/Acetonitrile (50/50) 112 A 136 A 160 A 184 A + HAcetonitrile/Chloroform (50/50) 113 A 137 A 161 A 185 A Heptane/Ethylformate (50/50) 114 A 138 A 162 A 186 A 1,4-Dioxane/Cyclohexane (50/50)115 A 139 A 163 A 187 A Water/Methanol (50/50) 116 A 140 A 164 A 188 ACyclohexane/N-Methylpyrrolidone (50/50) 117 A 141 A 165 A 189 ATetrahydrofuran/N-Methylpyrrolidone (50/50) 118 A + K 142 A 166 A 190 A→ A + K 1,2,3,4-Tetrahydronaphthalene/Acetonitrile (50/50) 119 A 143 A167 A 191 A Chlorobenzene/N-Methylpyrrolidone (50/50) 120 A 144 A 168 A192 A

TABLE 14 Cooling-evaporative crystallization experimental results: Tprofile 4 T profile 4 Conc. (mg/mL) 40 80 Solvent Expt. Form Expt. Formtert-Butyl methyl ether 193 A 217 A Methyl acetate 194 A 218 A Methanol195 — 219 A Tetrahydrofuran 196 A 220 A Acetonitrile 197 A 221 A1,2-Dimethoxyethane 198 A 222 A Isopropyl acetate 199 A 223 A1,4-Dioxane 200 A 224 A 2-Methoxyethanol 201 A 225 A 2-Hexanone 202 A226 A Heptane 203 A 227 A 1-Pentanol 204 A 228 A Acetone/Dichloromethane205 A 229 A (50/50) Methanol/Chloroform 206 H → 230 H → (50/50) A + H Atert-Butyl methyl 207 A 231 A ether/Chloroform (50/50)Methanol/Acetonitrile 208 A 232 A (50/50) Acetonitrile/Chloroform 209 A233 A (50/50) Heptane/Ethyl formate 210 A 234 — (50/50)1,4-Dioxane/Cyclohexane 211 A 235 A (50/50) Water/Methanol (50/50) 212 A236 A Cyclohexane/N- 213 A 237 A Methylpyrrolidone (50/50)Tetrahydrofuran/N- 214 A 238 A Methylpyrrolidone (50/50) 1,2,3,4- 215 A239 A Tetrahydronaphthalene/ Acetonitrile (50/50) Chlorobenzene/ 216 A240 A N-Methylpyrrolidone (50/50)

Evaporative Crystallization Experiments

Brigatinib Form A and 30 different solutions were employed. In a vial,20 mg of material was weighed and 1000 μL of the given solvent wasadded. After stirring at rt for a maximum of 3 hours, the solvents wereevaporated at rt (at 200 mbar for 120 h, then 5 mbar for 48 hours).Solids obtained were analyzed dry by XRPD and digital imaging as shownin Table 15.

TABLE 15 Evaporative crystallization experiments Mass Solvent Dissolved?Form (XRPD) 19.8 Acetone N A 21.4 Cyclohexane N A 21.7 Acetonitrile N A23.3 Isopropyl Acetate N A 19.4 n-Heptane N A 21.7 Cyclohexanone N A19.7 Ethyl formate N A 21.3 tert-Butyl methyl ether N A 20.6 ChloroformY A 19.8 Methanol Y A 21.8 Hexane N A 21.2 Ethyl acetate N A 20.3Ethanol N A 20.9 2-Butanone N A 21.6 Isopropanol N A 20.8 EthyleneGlycol Dimethyl Ether N A 21.4 2-Butanol N A 21.3 1,4-Dioxane N A 20.2Toluene N A 20.3 Butyl acetate N A 19.7 2-Hexanone N A 20.8 Anisole N A20 N,N-Dimethylacetamide N A 20.2 Dichloromethane Y A 20.8 Acetone/Water(50/50) N A + C 19.8 Cyclohexane/Tetrahydrofuran N A (50/50) 20.1Water/Methanol (50/50) N A 20.3 Cyclohexane/1,4-Dioxane (50/50) N A 20.4Water/Ethanol (50/50) Y A 20.5 Cyclohexane/Cyclohexanone(50/50) N A 20.32,2,4-Trimethylpentane/3,3- N A Dimethyl-2-butanone (50/50) 20.5Water/1,2-Propanediol N A 20.6 Water/Formamide N A 20.2Cyclohexanone/cis-Decalin N A

Vapor Exposure Experiments

The stability of Form A upon exposure to solvent vapors was investigatedin twenty solvents as shown in Table 16. Approximately 20 mg ofbrigatinib Form A was weighed in 1.8 mL vials. The vials were left openand placed in closed 40 mL vials containing 2 mL of solvent. Thematerial was exposed to solvent vapours at room temperature for twoweeks. At the end of the experiment time, the solids were harvested wetand dry and analyzed by XRPD and digital imaging.

TABLE 16 Vapor exposure experiments Form A weight (mg) Solvent Form 20.9Water A 21.5 Acetone A 20.8 Acetonitrile A 19.9 n-Heptane A 20.7Isopropyl Acetate A 20 2-Methyltetrahydrofuran A 21.5 Tetrahydrofuran A20 Methanol A 20.5 Ethanol A 20.8 Isopropanol A 19.5 Isobutanol A 19.6Methyl acetate A 19.6 Ethyl acetate A 21.1 Propyl acetate A 21.22-Butanone A 21.6 Ethyl Formate A 20.2 tert-Butyl methyl ether A 20.8cyclohexane A

Cooling Crystallization with Hot Filtration Experiments

The cooling crystallization method with hot filtration included 34solvents and solvent mixtures. Supersaturated solutions were prepared bystirring slurries of brigatinib in 1300 μL of a given solvent or mixtureat 60° C. for one hour. Subsequently, the liquids were separated fromthe solids by filtration. The solutions were placed in a Crystal16™instrument to undergo the following cooling profile. Samples were warmedto 60° C. and held for 60 min, then cooled at a rate of 1° C./hr untilreaching 5° C. The samples were then held at that temperature for 48hrs. In each experiment, precipitation was not observed at the end ofthe thermal profile. The solvents were evaporated, at 200 mbar for 104hours and at 5 mbar for 70 hours. In several cases, evaporation at 5mbar continued for about 400 hours while in some other cases, no yieldwas obtained after evaporation of the solvent. All obtained solids wereanalyzed by XRPD and digital imaging. Table 17 provides the appliedcrystallization conditions and corresponding obtained solid forms.

TABLE 17 Cooling crystallization with hot filtration experiments SlurrySolid conc. Slurry after (mg/ at thermal Form Solvent mL) 60° C.? cycle(XRPD) Methanol/Acetonitrile 86 N N A Acetone/Water 45 Y N A + CAcetonitrile/Chloroform 67 N N A Cyclohexane/Tetrahydrofuran 23 Y Y Atert-Butyl methyl ether/1,2- 31 Y N A Propanediol Isoamylacetate/Chloroform 46 N N A Isopropyl ether/Diethoxymethane 22 Y N —2,2,4-Trimethylpentane/Isopropyl 21 Y N — ether Water/Methanol 45 Y NA + C Cyclohexane/1,4-Dioxane 21 Y N A Water/Ethanol 131 Y N ACyclohexanone/Tetrahydrofuran 43 Y N A Water/1,4-Dioxane 66 Y Y A + CIsopropyl ether/ p-Xylene 19 Y N A Cyclohexane/Cyclohexanone 19 Y N A2,2,4-Trimethylpentane/Pinacolone 25 Y N Am Cyclohexane/cis-Decahydro-23 Y N — naphthalene Water/Isopropyl Acetate 22 Y N AWater/1,2-Propanediol 24 Y N A Water/Formamide 22 Y N —n-Heptane/p-Xylene 26 Y N — 2,2,4-Trimethylpentane/Mesitylene 24 N N —cis-Decahydronaphthalene/ 18 Y N — MethylCyclohexane2,2,4-Trimethylpentane/cis- 26 Y N — Decahydro-naphthalenep-Xylene/Anisole 23 Y N A n-Nonane/1-Octanol 22 Y N — n-Amylacetate/1-Octanol 20 Y N A 1,2,3,4-Tetrahydronaphthalene/ 21 Y N ACumene Cyclohexanone/cis- 23 Y N A DecahydronaphthaleneCumene/cis-Decahydronaphthalene 21 Y N — Anisole/Nitrobenzene 44 Y N ACyclohexanone/N-Methyl-2- 87 Y N A pyrrolidone Ethyleneglycoldiacetate/Bis(2- 25 Y N A methoxyethyl)ether Cyclohexanone/Nitrobenzene22 Y N A

Crash Crystallization with Anti-Solvent Addition

In the crash-crystallization experiments, 34 different crystallizationconditions were applied, using 6 different solvents and 24 differentanti-solvents (see Table 17). The anti-solvent addition experiments wereperformed forward. For each solvent, a stock solution was prepared, theconcentration of brigatinib in each case being that attained atsaturation at ambient temperature after equilibration for 24 hoursbefore filtering.

For each experiment, the anti-solvent was added to each solvent vial,with a solvent to anti-solvent ratio of 1:0.25. In the cases where noprecipitation occurred, this ratio was increased to 1:1, and if again noprecipitation occurred the ratio was increased to 1:4, with a waitingtime of 60 minutes between the additions (up to the third addition) and35 minutes between the third addition and fourth addition. When nocrystallization occurred or not enough solids precipitated forseparation, samples were kept at 5° C. for 17 hours. The precipitatedsolids were separated from the liquids by centrifugation anddecantation. When decantation could not be applied, the liquid wascarefully removed using Pasteur's pipettes. The solids were dried at 200mbar for 17 hours and analyzed by XRPD and digital imaging. In the caseswhere no precipitation occurred, the solvents were evaporated at 200mbar for 17 hours prior to lowering the vacuum to 5 mbar. All obtainedsolids were analyzed by XRPD and digital imaging. The measuring platescontaining the final solid were stored at ambient temperature for 5weeks. The solid form was assessed again by XRPD. The arrows in Table 18indicate if the form changed during storage.

TABLE 18 Crash crystallization with anti-solvent addition experimentsRatio S:AS Vol Forward Precip- Form Solvent (μL) Anti-solvent (1:x)itation (XRPD) Chloroform 150 tert-Butyl methyl 4 Yes A + G ether (wet),A (dry, ML*) Methanol 900 Acetonitrile 4 No A Acetone 7400 Water 4 No**— Chloroform 150 Acetonitrile 1 Yes G (wet) → A, A (dry, ML*)Cyclohexane 7400 Tetrahydrofuran 4 No** — tert-Butyl methyl 74001,2-Propane diol 4 No** — ether Diisopropyl ether 7400 Diethoxyme ane 4No** — 2,2,4- 7400 Isopropyl ether 4 No** — Trimethylpentane Methanol900 Water 4 No D**** 1,4-Dioxane 3900 Cyclohexane 4 No A Ethanol 4900Water 4 No A + H Tetrahydrofuran 3900 Cyclohexanone 4 No A n-Heptane7400 Cyclohexane 4 No** — 1,4-Dioxane 3900 Water 4 No A + H Cyclohexane7400 Cyclohexanone 4 No** — 3,3-Dimethyl-2- 7400 2,2,4- 1 Yes** —butanone Trimethylpentane Cyclohexane 7400 Cis-Decahydro- 1*** No** —naphthalene Isopropyl Acetate 7400 Water 4 No** — 1,2-Propanediol 7400Water 4 No** — Formamide 7400 Water 4 No** — n-Heptane, 7400 P-Xylene 4No** — Cis-Decahydro- 7400 Methylcyclo- 4 No** — naphthalene hexane2,2,4-Trimethyl 7400 Cis-Decahydro- 4*** No** — pentane naphthaleneAnisole 7400 P-Xylene 4 No A 1-Octanol 7400 n-Nonane 4*** No A 1-Octanol7400 N-Amyl acetate 4 No A 1,2,3,4-Tetrahydro 7400 Cumene 4 No** —naphthalene Cyclohexanone 7400 Cis-Decahydro- 4*** No A naphthaleneN-Amyl acetate 7400 Ethyleneglycol 4 No** — diacetate Cumene 7400Cis-Decahydro- 4*** No** — naphthalene Isoamyl acetate 7400 Nitrobenzene4 No** — Anisole 7400 Nitrobenzene 4 No** — Cyclohexanone 7400N-Methyl-2- 4 No** — pyrrolidone Ethylene glycol 7400 Bis(2-methoxy 4No** — diacetate ethyl) ether *ML = From mother liquour; **No yield;***Two additions applied; ****Single crystal picked from liquid

Slurry Experiments

A total of 68 slurry experiments were performed with brigatinib both atroom temperature (20° C.) and 40° C., using 34 solvents. In all cases, asolvent volume of 250 μL was used. The slurries were stirred for twoweeks. At the end of the slurry time, the vials were centrifuged andsolids and mother liquids separated. The solids were analyzed wet anddry by XRPD and digital imaging. The measuring plates were then storedat ambient conditions for 3-4 weeks and another XRPD was obtained of thesolid, any form change is shown by an arrow. Tables 19a and 19bsummarizes the experimental conditions and obtained solid forms

TABLE 19a Slurry experiments at 20° C. Mass Conc. Form wet Form dry (mg)Solvent (mg/mL) (XRPD) (XRPD) 22.6 Ethyl formate 90.4 A A 22.4tert-Butyl methyl 89.6 A A ether 26.3 Acetone 105.2 A A 23.8 Methylacetate 95.2 A A 22.6 Chloroform* 90.4 — A + E 19.5 Methanol 78 A A 23.9Tetrahydrofuran 95.6 A A 19.2 Hexane 76.8 A + L → A + L A + L → A + L19.9 Ethyl acetate 79.6 A A 20.5 Ethanol 82 A A 23.0 Cyclohexane 92 A A20.5 Acetonitrile 82 A A 20.9 2-Propanol 83.6 A A 24.0 1,2-Dimethoxy 96A A ethane 20.8 Isopropyl acetate 83.2 A A 20.0 Hepta e 80 A + L → A + LA 25.8 2-Butanol 103.2 A A 24.6 Water 98.4 A A 23.3 Methylcyclohexane93.2 A + L → A + L A 18.4 1,4-Dioxane 73.6 A A 18.6 N-propyl acetate74.4 A A 21.7 Isobutanol 86.8 A A 23.9 Toluene 95.6 A A 24.0 Isobutylacta e 96 A A 23.3 2-Methoxyethanol 93.2 A A 24.9 n-Butyl acetate 99.6 A A26.6 2-Hexanone 106.4 A A 19.1 Chlorobenzene 76.4 A A 18.92-Ethoxyethanol 75.6 A A 24.8 1-Pentanol 99.2 A A 21.2 m-Xylene 84.8 A A19.7 Cumene 78.8 A A 23.2 N,N-Dimethyl 92.8 A A formamide 18.5 Anisole74 A A *in this experiment, the solids dissolved after 14 days

TABLE 19b Slurry experiments at 40° C. Mass Form wet Form dry (mg)Solvent Conc. (mg/mL) (XRPD) (XRPD) 33.8 Ethyl formate 135.2 A A 33.9tert-Butyl methyl 135.6 A A ether 35.8 Acetone 143.2 A A 34.9 Methylacetate 139.6 A A 35.9 Chloroform* 143.6 — A + E 33.3 Methanol 133.2 A A37.6 Tetrahydrofuran 150.4 A A 33.6 Hexane 134.4 A A 31.6 Ethyl acetate126.4 A A 33.2 Ethanol 132.8 A A 31.5 Cyclohexane 126 A A 36.5Acetonitrile 146 A A 35.9 2-Propanol 143.6 A A 37.7 1,2-Dimethoxy 150.8A A ethane 37.1 Isopropyl acetate 148.4 A A 32.9 Heptane 131.6 A A 41.32-Butanol 165.2 A A 32.2 Water 128.8 A A 32.0 Methylcyclohexane 128 A A36.4 1,4-Dioxane 145.6 A A 37.9 N-propyl acetate 151.6 A A 36.1Isobutanol 144.4 A A 30.3 Toluene 121.2 A A 33.7 Isobutylacetate 134.8 AA 31.0 2-Methoxyethanol 124 A A 34.1 n-Butyl acetate 136.4 A A 33.52-Hexanone 134 A A 35.9 Chlorobenzene 143.6 A A 33.2 2-Ethoxyethanol132.8 A A 39.2 1-Pentanol 156.8 A A 33.2 m-Xylene 132.8 A A 41.1 Cumene164.4 A A 34.1 N,N-Dimethyl 136.4 A A formamide 33.3 Anisole 133.2 A A*in this experiment, the solids dissolved after 14 days

In a second set of slurry experiments, the same amounts of Form A andForm B were weighed into 1.8 mL vials, and charged with a stirring bar.After addition of the solvent, the slurries were placed at 25° C. and50° C., under stirring. Material from the slurries was sampled at thetime points of 2, 4 and 14 days (sampling from the same vial per solventand per temperature). These materials were analyzed wet by XRPD anddigital imaging. As seen in Table 20, Form B converted to Form A in allorganic solvents and in water at 37° C. The sampling after 2 and 4 daysin water at 25° C. showed that the solids were a mixture of Form A andthe hepta-hydrated Form D. This observation indicated that Form Bconverted to Form D in an aqueous environment and that Form A remainedstable. In the sampling on the 14th day, only Form A was present,indicating its higher stability in water, compared to Form D.

TABLE 20 Slurry experiments 2 Days 4 days 2 weeks Weight (mg) Form A/BSolvent 25° C. 60° C. 25° C. 60° C. 25° C. 60° C.   15 A Water A + D AA + D A A A 16.5 A n-Heptane A A A A A A   17 A 1-Butanol A A A A A A  18 A Methanol A A A A A A 18.5 A Acetone A A A A A A 28.7 B Water A AA A A A 34.7 B n-Heptane A A A A A A 27.4 B 1-Butanol A A A A A A 28.4 BMethanol A A A A A A 27.6 B Acetone A A A A A A

Vapor Diffusion into Solution Experiments

For the vapor diffusion into solution experiments, saturated solutionsof brigatinib were exposed to anti-solvent vapors at room temperaturefor two weeks. An aliquot of saturated solution was transferred to avial which was left open and placed in a closed container with antisolvent (see Table 20). After two weeks, the samples were evaluated forsolid formation. Where solids were present, the liquids were separatedfrom the solids, which were then dried at full vacuum. In the caseswhere no precipitation was observed, the solvents were placed overnightat 5° C. to promote precipitation. If no solids were present, theliquids were evaporated at 200 mbar for 75 hours, or, if still no solidswere present, the liquids were further evaporated at 10 mbar for amaximum of 10 days. All obtained solids were analyzed dry by XRPD anddigital imaging. Table 21 provides the experimental conditions andcorresponding solid forms obtained.

TABLE 21 Vapor diffusion into solution experiments Volume Solid afterForm Solvent (μL) Anti-solvent 2 weeks? (XRPD) Anisole 8000 NitrobenzeneN A P-Xylene 30000 Anisole N A Diisopropyl 5000 Diethoxymethane N —ether* Isopropyl 40000 Water N A Acetate Cyclohexanone 40000Cis-Decahydro- N A naphthalene Cyclohexanone 40000 N-Methyl-2- N Apyrrolidone Ethyl Formate 8000 n-Hexane N A Ethyl Formate 8000Cyclohexane N A Ethyl Formate 8000 2,2,4- N A Trimethylpentane EthylFormate 8000 n-Heptane N A Tetrahydrofuran 40000 Cyclohexanone N AEthylene Glycol 8000 n-Pentane N A Dimethyl Ether Ethylene Glycol 80002-Methylpentane N A Dimethyl Ether Ethylene Glycol 8000 n-Hexane N ADimethyl Ether Ethylene Glycol 8000 Cyclohexane N A Dimethyl EtherEthylene Glycol 8000 n-Heptane N A Dimethyl Ether Ethylene glycol 8000Bis(2-methoxyethyl) N — diacetate* ether n-Nonane* 40000 1-Octanol N —1,2,3,4- 8000 Cumene N — Tetrahydro naphthalene* Dioxane, 1,4- 2000Cyclohexane N A (Extra dry) Isoamyl acetate* 4000 Nitrobenzene N An-Heptane 40000 P-Xylene N A Cis-Decahydro- 8000 MethylCyclohexane N Amnaphthalene 2,2,4-Trimethyl 8000 Cis-Decahydro- N A pentane naphthalene1,2-Propanediol* 8000 tert-Butylmethyl N — ether 1,2-Propanediol* 8000Water N — N-Amyl acetate 8000 1-Octanol N A N-Amyl acetate* 8000Ethyleneglycol N — diacetate Ethanol 2000 Water N A Methanol 600 Water YA Acetone* 8000 Water N — Chloroform* 200 tert-Butylmethyl N — etherChloroform* 200 Acetonitrile Y — Cumene 8000 Cis-Decahydro- N Anaphthalene *No yield after evaporation

Vapor Diffusion Onto Solids Experiments

For the 34 vapor diffusion onto solids experiments, amorphous brigatinibwas prepared by grinding the starting material for 4 hours. The vialscontaining the amorphous brigatinib were left open and placed in closed40 mL vials containing 2 mL of solvent (see Table 21). The amorphousbrigatinib was exposed to solvent vapors at room temperature for twoweeks. At the end of the experiment time, the solids were harvested wetand dry and analyzed by XRPD and digital imaging. For the appliedcrystallization conditions and corresponding obtained solid forms seeTable 22.

TABLE 22 Vapor diffusion onto solids Solids after 2 Form wet Form drySolvent Weight (mg) weeks? (XRPD) (XRPD) Ethyl ether 30.7 Y A AN-pentane 34.8 Y A A Dichloromethane 30.1 N — Am Ethyl formate 29.8 Y AA tert-Butylmethyl 30.5 Y A A ether Acetone 33.7 Y A A Methyl acetate31.2 Y A A Chloroform 28.6 N — A + Am Methanol 27.6 Y A ATetrahydrofuran 31.7 Y A A Hexane 29.3 Y A A Ethyl acetate 35.9 Y A AEthanol 30.7 Y A A 2-Butanone 28.8 Y A A Cyclohexane 29.2 Y A AAcetonitrile 29.1 Y A A 2-Propanol 29.8 Y A A 1,2-Dimethoxy 36.1 Y A Aethane Isopropyl acetate 30.3 Y A A 1-Propanol 30.1 Y A A Heptane 39.1 YA A 2-Butanol 29.7 Y A A MethylCyclohexane 29.9 Y A A N-propyl acetate34.8 Y A A 1,4-Dioxane 35.1 Y A A Isobutanol 31.3 Y A A Toluene 37.5 Y AA Isobutylacetate 33.1 Y A A 1-Butanol 38.1 Y A A Water 37.6 Y A A + B +C n-Butyl acetate 35.4 Y A A 2-Hexanone 31.9 Y A A Chlorobenzene 33.8 YA A 2-Ethoxyethanol 32.0 Y A A

Solvent Assisted Grinding Experiments

In the solvent assisted grinding experiments, a small amount of solventwas added to solid brigatinib which had been mechanically ground in astainless steel vial containing 2 stainless steel grinding balls. Inthis manner, 17 different solvents were investigated. Typically, 30 mgof starting material was weighed into the grinding vial and 10 μL ofsolvent was added to the vial. The grinding experiments were performedat 30 Hz for 60 min. Subsequently, the samples were collected andanalyzed (wet) by XRPD and digital imaging. For the appliedcrystallization conditions and corresponding obtained solid forms seeTable 23.

TABLE 23 Solvent assisted grinding experiments Weight Volume FormSolvent (mg) (μL) (XRPD) Ethanol 30.7 10 A Cyclohexane 30.8 10 AAcetonitrile 34.1 10 A 2-propanol 35.0 10 A Ethylene Glycol 31.5 10 ADimethyl Ether Isopropyl Acetate 30.3 10 A n-Heptane 32.1 10 A Water32.5 10 A 1,4-Dioxane 32.0 10 A Isobutanol 31.5 10 A Toluene 31.8 10 AButyl acetate 33.0 10 A 2-Hexanone 30.7 10 A Chlorobenzene 30.8 10 AAcetone 30.3 10 A Cumene 31.0 10 A Anisole 31.8 10 A

Thermocycling Experiments

A total of 33 slurries and 1 solution (chloroform) of starting materialin solvents were prepared at room temperature. The mixtures were placedin a Crystal16™ to undergo the following temperature profile:

1. Heating at a rate of 5° C./h until reaching 40° C., with stirring(500 rpm)

2. Cooling at a rate of 5° C./h until 5° C., with stirring (200 rpm)

3. Aging for 30 min at 5° C.

4. Repeat 8 cycles

After completion of the cycling program, the solids were separated fromthe mother liquids by centrifugation, dried under 200 mbar for 48 hours(2-ethoxyethanol for 283 hours) and analyzed by XRPD and digitalimaging. For the applied crystallization conditions and correspondingobtained solid forms, see Table 24. Solid form (or mixture) followingthe arrow (→) was obtained upon remeasurement by XRPD after storage ofthe measuring plates at ambient conditions for 5 weeks.

TABLE 24 Thermocycling experiments Vol- Solids Weight ume after lastForm dry Form mother Solvent (mg) (μL) cycle? (XRPD) liquid (XRPD) EthylFormate 20.5 750 Y A — tert-Butylmethyl 20.1 750 Y A — ether Acetone20.0 750 Y A — Methyl acetate 21.9 750 Y A — Chloroform 173.3 400 Y AA + G Methanol 20.6 500 N A — Tetrahydrofuran 20.6 750 Y A A Hexane 20.4750 Y A — Ethyl acetate 22.0 750 Y A — Ethanol 19.2 750 Y A ACyclohexane 19.8 750 Y A — Acetonitrile 19.8 750 Y A — Isopropanol 21.4750 Y A — Ethylene Glycol 23.4 750 Y A — Dimethyl Ether IsopropylAcetate 20.0 750 Y A — n-Heptane 19.2 750 Y A A + L → A 2-Butanol 17.9750 Y A — Water 21.7 750 Y A — MethylCyclo- 18.9 750 Y A — hexane1,4-Dioxane 21.7 750 Y A — Propyl acetate 23.7 750 Y A — Isobutanol 21.3750 Y A A Toluene 20.5 750 Y A — lsobutyl aceta 21.0 750 Y A —2-Methoxyethanol 56.5 750 Y A A Butyl acetate 18.9 750 Y A — 2-Hexanone22.1 750 Y A — Chlorobenzene 20.0 750 Y A A 2-Ethoxyethanol 20.1 750 N A— 1-Pentanol 19.4 750 Y A A m-Xylene 20.4 750 Y A — Cumene 19.7 750 Y A— N,N-Dimethyl 20.0 750 Y A A formamide Anisole 18.8 750 Y A AVariable Temperature XRPD Experiments

Data was collected for Forms A, B, C, and D almost immediately afterreaching the target temperature (within approximately 10 min).

For Form A, the temperatures used in the experiment were 25, 40, 60,100, 120, 140, 150, 160, 170, 180, 190, and 200° C. Data collectionlasted 20 min per temperature and the stabilization time in between was10 min. The variable temperature XRPD data collected for Form A did notreveal any phase transformation. The only peak shifts observed wereattributed to thermal expansion.

For Form B, the temperatures used in the experiment were 25, 40, 60,100, 120, 140, 150, 155, 160, 165, 170, 180, and 190° C. Data collectionlasted 45 min per temperature and the stabilization time in between was10 min. At 150° C., partial conversion to Form A was observed and at155° C. the conversion was complete. Thereafter, Form A remained stablefor the rest of the temperature profile.

For Form C, the temperatures used in the experiment were 25, 40, 60, 70,80, 100, 120, 140, 150, 155, 160, 165, 170, 175, 180, 190, and 200° C.Data collection lasted 40 min per temperature and the stabilization timein between was 10 min. Form C is instable at temperatures higher than25° C. By the first measurement, the material had already partiallyconverted to the dehydrated Form B. Thereafter, the solid formtransformations resembled those observed in the VT-XRPD experiments ofForm B, with the difference that the transformation of Form B to Form Awas initiated already at 120° C. The conversion was completed though atthe same temperature (155° C.). Again, no phase transition was observedupon cooling.

For Form D, the temperatures used in the experiment were 25, 35, 45, 55,65, 75, 85, 100, 120, 140, 150, 155, 160, 165, 170, 175, 180, 190, and200° C. Data collection lasted 10 min for temperatures 25-85° C. with astabilization time of 1 min, and 40 min for temperatures 100-25° C. witha stabilization time of 10 min. The variable temperature XRPD confirmedthat the hepta-hydrated Form D is unstable at temperatures higher than25° C. Indeed, after the first measurement, Form D had already converted(partially) to the hydrated Form C (at 35° C.) and to the dehydratedForm B at 45° C. Thereafter, the solid form transformations resembledthose observed in the VT-XRPD experiments of Form C: Form B converted(partially) to Form A at 120° C. The conversion was completed at 150° C.No phase transition was observed upon cooling.

Variable Humidity-XRPD Experiments

The relative humidity was increased from the starting amount, brought upto the maximum, then dried back to the minimum value. The datacollection time was 41 min at each step, time starting afterequilibration of the relative humidity.

For Form A, the collected XRPD patterns of Form A did not show any phasetransition, neither at 30 nor at 60° C. Only some minor, but clear, peakshifts of the order of 0.03°2θ were observed in specific peaks, startingat about 60% RH. The peaks shifts were reversible at RH of about 30%. Asample of Form A was then exposed to 80% RH for 15 h. The peaks shiftshad occurred after 90 min, and the extent of the shifts remainedconstant throughout the exposure at 80% RH for 15 h. Upon return to 10%RH, the peaks shifted to their original position. To investigate theamount of water adsorbed, a new sample of Form A was exposed for 2 h at80% and a TGMS of this sample was measured. The TGMS thermogram showed amass loss of 0.35% corresponding to 0.1 water molecules.

For Form B, the relative humidity was measured at 30° C. The RH % valuesmeasured were 10, 30, 50, 60, 65, 70, 75, and 80%. Upon sorption, Form Bconverts to the hydrated Form C, starting at about 65% RH. At 80% RH theconversion to Form C was completed. Upon desorption, Form C dehydratesto Form B, starting at about 30% RH. At 10% RH the conversion to Form Bwas completed.

For Form C, a hydrate, the relative humidity was measured at 30° C. Theexperiment was performed starting at the maximum RH and dehydrated, thenrehydrated back up to the maximum value. The RH % values measured were10, 15, 20, 25, 30, 35, 40, 60, and 80%. Upon desorption, Form Cdehydrated to Form B, starting at about 25% RH. At 10% RH, theconversion to Form B was complete. Upon sorption, Form B converted toForm C, starting at about 60% RH. At 80% RH the conversion to Form C wascomplete. The results are consistent with the corresponding experimentsof Form B.

For Form D, a hydrate, the relative humidity was measured at 30° C. Theexperiment was performed starting at the maximum RH and dehydrated, thenrehydrated back up to the maximum value. The RH % values measured were10, 15, 20, 25, 30, 35, 40, 60, and 80%. Despite attempts to havefreshly prepared Form D, even the first measurements at 80% RH showedthat the solid had already partially transformed to Form C. Thereafter,the solid transformed to the hydrated Form C and eventually to theanhydrous Form B, as already observed in the VH-XRPD measurements ofForms B and C. Upon desorption, the hepta-hydrated Form D converted tothe hydrated Form C. Form C dehydrated to Form B, starting at about 20%RH. At 10% RH, the conversion to Form B was complete. Upon sorption,Form B converted to Form C, starting at about 40% RH. At 80% RH, theconversion to Form C was complete. The solid did not hydrate to Form Das a relative humidity of 80% is not sufficient; for the conversion toForm D, an exposure at relative humidity of 95% can be employed.

Dynamic Vapor Sorption Experiments

In three DVS experiments, the relative humidity was varied as follows:

Expt. 1: 5%→95%→65% RH

Expt. 2: 5%→95%→5% RH

Expt. 3: 5%→95% RH

Expt. 4: 0% for 6 h→5% for 1 h→15% for 1 h→25-85% gradient over 2 h→95for 5 h RH

For Expt. 1, during sorption, Form B adsorbed water mass correspondingto 2.26 molecules of water between 45-95% RH, as shown in FIG. 44. Upondesorption to 65% RH, the gained water mass remained almost constant.XRPD measurement of the solid showed that it was the hydrated Form C.The additional gained water mass can be attributed to adsorption on thesurface of the material.

For Expt. 2, during sorption, a two-step water mass gain was observed,as shown in FIG. 45. In the first step, between 45-85% RH, a mass changeof 6.45% was observed corresponding to 2.1 water molecules. The data areconsistent with the hydrated Form C being formed at this stage. In thesecond step, between 85% and 95% RH, a total change in mass of 16.7% wasreached. A further mass increase of 17.4% was observed at 85% RH duringdesorption. The increasing mass gain during desorption indicates that noequilibrium was reached within one hour at 95% RH, and the wateradsorption continued at least until 85% RH, during humidity decrease.The maximum change in mass corresponded to 5.6 water molecules. The dataare consistent with the hepta-hydrated Form D being (partially) formedat the maximum RH. During the two-step desorption, the change in masswas roughly stable up to about 75% RH and it, thereafter, reduced toabout 5.2%. The latter change in mass corresponded to about 1.7 watermolecules. At this stage, the data was consistent with the hydrated FormC being formed. Thereafter, and until about 25% RH, the gained massdecreased to 4.2%, corresponding to 1.4 water molecules. The data isconsistent with a mixture of the hydrated Form C with the anhydrous FormB being formed. Thereafter, the gained water was lost in one step,between 25% and 15% RH. The XRPD of the material at the end of thesorption-desorption cycle showed that it was a mixture of Forms B and C.

For Expt. 3, the DVS indicated a two-step water adsorption as shown inFIG. 46. The change in mass during the first step (between 45-85% RH)was 5.59% corresponding to 1.8 water molecules. The total change in massat 95% RH was 15.88% corresponding to 5.15 water molecules. XRPDmeasurement of the solid after the cycle showed that it was Forms B+C.

For Expt. 4, deviations between the measured values of water mass gainor losses and the expected corresponding water molecules can beattributed to the fact that the measurements were performed prior toreaching the equilibrium of an event. Therefore, in this experiment, theRelative Humidity profile was modified in order to investigate theimpact of longer equilibration time at each step. As seen in FIG. 47,the maximum change in mass was 22.2% corresponding to 7.2 watermolecules. The XRPD pattern of the material after the cycle was FormsC+D.

Hydration Studies of Forms A and B

Slurrying of Forms A and B (separately) was performed at roomtemperature in water, HCl buffer of pH 1.0 (0.1N HCl) and SGF (for FormA). The solids were harvested and measured wet by XRPD after 45 min, 1.5h, 15 h, 48 h and 10 days (not in SGF). Form A remained stable evenafter 10 days slurrying in water and the HCl buffer or 1.5 h in SGF.Form B converted to the heptahydrate Form D after 45 min, which remainedstable, at least for 10 days. In a separate experiment, where Form B wasexposed to 90% RH for one day, the material converted to a mixture ofForms C and D.

Dehydration of Forms C and D

In Table 25, a list of drying conditions for Form C are presentedtogether with the final solid form. At ambient pressure, Form C appearsto be stable after 1.5 hours at 30° C. while at 40° C. it converted toForm B within one hour.

In Table 26, a list of drying processes of Form D are presented. Form Dunder 5 mbar pressure and at 60° C. led to the formation of Form B after24 h. In some instances, small quantities of Form C were visible on theXRPD patterns, even after 5 days of drying. This observation could beattributed to different particle morphology (fine particles vs.agglomerates/aggregates). At 60° C. and at 50 mbar pressure, Form Dconverted to a mixture of Forms B+C after 86 h and to Form B after 110h. In general, depending on the time and pressure, Forms B and C occur.

TABLE 25 Dehydration of Form C. Temp (° C.) 0.5 h 1 h 1.5 h 4.5 h 20 C —C — 30 C — C — 40 — B — B

TABLE 26 Dehydration of Form D Time (h) at 50 ambient 60° C. mbar 5 mbarpressure 24 — B — 28 — B + C — 5 days — B + C — 6 days — B — 15 (RT) — C— 20 (RT) — B + C — 15 — C + D — 20 — B + C — 86 B + C — — 110 B — — 65B + C — — 86 B — — 8 days, — — B + C closed vial at RT

In Table 27, the occurrence of solid forms of brigatinib is given,together with the crystallization methods from which they crystallizedand the related solvents. The table provides the results of over 600experiments with solid forms measured by XRPD wet and/or dry (wet anddry count as separate experiments). In eight cases, no form assignmentwas made due to low yield. The solid form(s) following the arrowwas/were obtained upon remeasurement by XRPD after storage of themeasuring plates at ambient conditions for several weeks (2-5 weeks).

TABLE 27 Summary of Brigatinib Solid Forms Obtained Form OccurrenceCrystallization Methods Solvent, Anti-Solvent Am 3 Hot filtration, 1;2,2,4-trimethylpentane/ Vapor diffusion onto solids, 1; Pinacolone(50/50); Vapor dissusion onto liquids, 1 Cis-Decahydro- naphthalene/Methylcyclohexane (50/50); Dichloromethane A 562 All methods Allsolvents D 1 Anti-solvent Methanol (S)/water(AS) E 1 Freeze-dryingChloroform F 1 Freeze-drying Trifluoroethanol/water (90:10) G → A 1Anti-solvent Chloroform (S) / acetonitrile (AS) H → 5Cooling-evaporative (μL scale) Methanol/Chloroform A + H, A (50/50) A +Am 1 Vapor diffusion on onto Chloroform solids A + B + C 1 Vapordiffusion onto solids Water A + C 4 Hot filtration, 3 Acetone/Water(50/50) Evaporative, 1 Water/Methanol (50/50) Water/1,4-Dioxane (50/50)Acetone/Water (50/50) A + E 2 Slurry Chloroform A + G 2 Anti-solvent, 1Chloroform (S)/ Thermocycling, 1 tert-Butyl methyl ether (AS) ChloroformA + H 12 AS, 2 Ethanol (S)/Water(AS) Cooling-evaporative (μL scale),1,4-Dioxane(S)/Water(AS) 10 Methanol Methanol/ Chloroform (50/50)Methanol/Acetonitrile (50/50) A + J → 1 Cooling-evaporative (μL scale)2-Methoxyethanol A + J A + K → 1 Cooling-evaporative (μL scale)Tetrahydrofuran/N- A + K Methyl-2-pyrrolidone (50/50) A + L → 4 SlurryHexane; A +L, A n-Heptane; MethylcyclohexaneIII. Pharmaceutical Compositions

In some embodiments, the present disclosure provides pharmaceuticalcompositions comprising at least one crystalline form of brigatinib andat least one component chosen from pharmaceutically acceptable carriers,pharmaceutically acceptable vehicles, and pharmaceutically acceptableexcipients. In some embodiments, the at least one crystalline form ofbrigatinib is present in a therapeutically effective amount. In someembodiments, the at least one crystalline form of brigatinib issubstantially pure. In some embodiments, the at least one crystallineform of brigatinib is chosen from Form A, Form B, Form C, Form D, FormE, Form F, Form G, and Form H. In some embodiments, the crystallinebrigatinib is Form A.

In some embodiments, a unit dosage form of a pharmaceutical compositioncomprises a single crystal form of brigatinib as the API. In someembodiments, the present disclosure provides pharmaceutical compositionsconsisting of one crystalline form of brigatinib. In some embodiments,the present disclosure provides pharmaceutical compositions consistingof one crystalline form of brigatinib and at least one component chosenfrom pharmaceutically acceptable carriers, pharmaceutically acceptablevehicles, and pharmaceutically acceptable excipients. In someembodiments, the present disclosure provides pharmaceutical compositionsconsisting essentially of one crystalline form of brigatinib andoptionally at least one component chosen from pharmaceuticallyacceptable carriers, pharmaceutically acceptable vehicles, andpharmaceutically acceptable excipients.

In some embodiments, the present disclosure provides pharmaceuticalcompositions produced by combining at least one crystalline form ofbrigatinib and at least one component chosen from pharmaceuticallyacceptable carriers, pharmaceutically acceptable vehicles, andpharmaceutically acceptable excipients.

In some embodiments, a unit dosage form of a pharmaceutical compositioncomprises more than one crystal form of brigatinib. In some embodiments,more than 50%, more than 70%, more than 80%, more than 90%, more than95%, or more than 99%, of brigatinib in the composition is in a singlecrystalline form. In some embodiments, the single crystalline form ofbrigatinib is chosen from Form A, Form B, Form C, Form D, Form E, FormF, Form G, and Form H. In some embodiments, the single crystalline formof brigatinib is Form A.

In some embodiments, one or all of the crystalline forms issubstantially pure. For example, in some embodiments, the pharmaceuticalcomposition comprises substantially pure Form A of brigatinib and atleast one component chosen from pharmaceutically acceptable carriers,pharmaceutically acceptable vehicles, and pharmaceutically acceptableexcipients. In some embodiments, a pharmaceutical composition comprisesForm A and Form B of brigatinib and at least one component chosen frompharmaceutically acceptable carriers, pharmaceutically acceptablevehicles, and pharmaceutically acceptable excipients. Other embodimentsare variations of this theme that will be readily apparent to those ofordinary skill in the art reading this disclosure. For example, in someembodiments, a pharmaceutical composition can comprise Form A and atleast one additional crystalline form of brigatinib chosen from Forms B,C, D, E, F, G, H, J, and K, and at least one component chosen frompharmaceutically acceptable carriers, pharmaceutically acceptablevehicles, and pharmaceutically acceptable excipients.

The at least one component may be readily chosen by one of ordinaryskill in the art and may be determined by the mode of administration.Illustrative and non-limiting examples of suitable modes ofadministration include oral, nasal, parenteral, topical, transdermal,and rectal. The pharmaceutical compositions disclosed herein can takeany pharmaceutical form recognizable to the skilled artisan as beingsuitable. Non-limiting examples of suitable pharmaceutical forms includesolid, semisolid, liquid, and lyophilized formulations, such as tablets,powders, capsules, suppositories, suspensions, liposomes, and aerosols.

in some embodiments, the pharmaceutical compositions optionally furthercomprise at least one additional therapeutic agent. In some embodiments,a compound as disclosed herein can be administered to a subjectundergoing one or more other therapeutic interventions (e.g. Crizotinibor other kinase inhibitors, interferon, bone marrow transplant, farnesyltransferase inhibitors, bisphosphonates, thalidomide, cancer vaccines,hormonal therapy, antibodies, radiation, etc). For example, in someembodiments, the compound as disclosed herein can be used as a componentof a combination therapy with at least one additional therapeutic agent(such as, for example, an anticancer agent), the at least one additionaltherapeutic agent being formulated together with or separately from thecompound as disclosed herein.

As used herein, the term “compound as disclosed herein” refers to atleast one crystalline form of brigatinib chosen from those disclosedherein, namely Forms A, B, C, D, E, F, G, H, J, and K, and amorphousbrigatinib. A compound as disclosed herein can be present in apharmaceutical composition as the single active agent or can be combinedwith at least one additional active agent which may be another form oramorphous brigatinib, or another non-brigatinib compound.

In some embodiments, a pharmaceutical composition disclosed herein canbe specially formulated for administration in solid or liquid form,including as non-limiting examples those adapted for the following: oraladministration, for example, drenches (aqueous or non-aqueous solutionsor suspensions), tablets (e.g., those targeted for buccal, sublingual,and systemic absorption), capsules, boluses, powders, granules, pastesfor application to the tongue, and intraduodenal routes; parenteraladministration, including intravenous, intraarterial, subcutaneous,intramuscular, intravascular, intraperitoneal or infusion as, forexample, a sterile solution, a sterile suspension, or asustained-release formulation; topical application, for example, as acream, an ointment, a controlled-release patch, or spray applied to theskin; intravaginally or intrarectally, for example, as a pessary, cream,stent or foam; sublingually; ocularly; pulmonarily; local delivery bycatheter or stent; intrathecally, or nasally.

Non-limiting examples of suitable carriers that can be employed inpharmaceutical compositions disclosed herein include water, ethanol,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), vegetable oils (such as olive oil), injectable organic esters(such as ethyl oleate) and mixtures thereof. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

In some embodiments, the compositions disclosed herein also comprise atleast one adjuvant chosen from preservatives, wetting agents,emulsifying agents, dispersing agents, lubricants, antioxidants,antibacterial agents, antifungal agents (e.g., paraben, chlorobutanol,phenol sorbic acid, and the like), isotonic agents (e.g., sugars, sodiumchloride, and the like), and agents capable of delaying absorption(e.g., aluminum monostearate, gelatin, and the like).

Methods of preparing the compositions disclosed herein may, for example,comprise bringing into association at least one compound as disclosedherein and other component(s), such as, for example, chemotherapeuticagent(s) and/or carrier(s). In some embodiments, the compositions areprepared by uniformly and intimately bringing into association acompound as disclosed herein with at least one carrier chosen fromliquid carriers and finely divided solid carriers, and then, ifnecessary, shaping the product.

Preparations for such pharmaceutical compositions are well-known in theart. See, e.g., Anderson, Philip O.; Knoben, James E.; Troutman, WilliamG, eds., Handbook of Clinical Drug Data, Tenth Edition, McGraw-Hill,2002; Pratt and Taylor, eds., Principles of Drug Action, Third Edition,Churchill Livingston, N.Y., 1990; Katzung, ed., Basic and ClinicalPharmacology, Ninth Edition, McGraw Hill, 2003; Goodman and Gilman,eds., The Pharmacological Basis of Therapeutics, Tenth Edition, McGrawHill, 2001; Remington's Pharmaceutical Sciences, 20th Ed., LippincottWilliams & Wilkins., 2000; Martindale, The Extra Pharmacopoeia,Thirty-Second Edition (The Pharmaceutical Press, London, 1999); all ofwhich are incorporated by reference herein in their entirety. Exceptinsofar as any conventional excipient medium is incompatible with thecompounds provided herein, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutically acceptable composition,the excipient's use is contemplated to be within the scope of thisdisclosure.

In some embodiments, the concentration of brigatinib in the disclosedpharmaceutical compositions is less than 100%, about 90%, about 80%,about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%,about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%,about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, about0.4%, about 0.3%, about 0.2%, about 0.1%, about 0.09%, about 0.08%,about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about0.02%, about 0.01%, about 0.009%, about 0.008%, about 0.007%, about0.006%, about 0.005%, about 0.004%, about 0.003%, about 0.002%, about0.001%, about 0.0009%, about 0.0008%, about 0.0007%, about 0.0006%,about 0.0005%, about 0.0004%, about 0.0003%, about 0.0002%, or about0.0001% w/w, w/v or v/v. As used herein, “about” means ±10% of the valuebeing modified.

In some embodiments, the concentration of brigatinib in the disclosedpharmaceutical compositions is greater than about 90%, about 80%, about70%, about 60%, about 50%, about 40%, about 30%, about 20%, about19.75%, about 19.50%, about 19.25% about 19%, about 18.75%, about18.50%, about 18.25%, about 18%, about 17.75%, about 17.50%, about17.25%, about 17%, about 16.75%, about 16.50%, about 16.25%, about 16%,about 15.75%, about 15.50%, about 15.25%, about 15%, about 14.75%, about14.50%, about 14.25%, about 14%, about 13.75%, about 13.50%, about13.25%, about 13%, about 12.75%, about 12.50%, about 12.25%, about 12%,about 11.75%, about 11.50%, about 11.25%, about 11%, about 10.75%, about10.50%, about 10.25%, about 10%, about 9.75%, about 9.50%, about 9.25%,about 9%, about 8.75%, about 8.50%, about 8.25%, about 8%, about 7.75%,about 7.50%, about 7.25%, about 7%, about 6.75%, about 6.50%, about6.25%, about 6%, about 5.75%, about 5.50%, about 5.25%, about 5%, about4.75%, about 4.50%, about 4.25%, about 4%, about 3.75%, about 3.50%,about 3.25%, about 3%, about 2.75%, about 2.50%, about 2.25%, about 2%,about 1.75%, about 1.50%, about 1.25%, about 1%, about 0.5%, about 0.4%,about 0.3%, about 0.2%, about 0.1%, about 0.09%, about 0.08%, about0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%,about 0.01%, about 0.009%, about 0.008%, about 0.007%, about 0.006%,about 0.005%, about 0.004%, about 0.003%, about 0.002%, about 0.001%,about 0.0009%, about 0.0008%, about 0.0007%, about 0.0006%, about0.0005%, about 0.0004%, about 0.0003%, about 0.0002%, or about 0.0001%w/w, w/v, or v/v. As used herein, “about” means ±10% of the value beingmodified.

In some embodiments, the concentration of brigatinib in the disclosedpharmaceutical compositions is ranges from approximately 0.0001% toapproximately 50%, approximately 0.001% to approximately 40%,approximately 0.01% to approximately 30%, approximately 0.02% toapproximately 29%, approximately 0.03% to approximately 28%,approximately 0.04% to approximately 27%, approximately 0.05% toapproximately 26%, approximately 0.06% to approximately 25%,approximately 0.07% to approximately 24%, approximately 0.08% toapproximately 23%, approximately 0.09% to approximately 22%,approximately 0.1% to approximately 21%, approximately 0.2% toapproximately 20%, approximately 0.3% to approximately 19%,approximately 0.4% to approximately 18%, approximately 0.5% toapproximately 17%, approximately 0.6% to approximately 16%,approximately 0.7% to approximately 15%, approximately 0.8% toapproximately 14%, approximately 0.9% to approximately 12%,approximately 1% to approximately 10% w/w, w/v or v/v, v/v. As usedherein, “approximately” means ±10% of the value being modified.

In some embodiments, the concentration of brigatinib in the disclosedpharmaceutical compositions ranges from approximately 0.001% toapproximately 10%, approximately 0.01% to approximately 5%,approximately 0.02% to approximately 4.5%, approximately 0.03% toapproximately 4%, approximately 0.04% to approximately 3.5%,approximately 0.05% to approximately 3%, approximately 0.06% toapproximately 2.5%, approximately 0.07% to approximately 2%,approximately 0.08% to approximately 1.5%, approximately 0.09% toapproximately 1%, approximately 0.1% to approximately 0.9% w/w, w/v orv/v. As used herein, “approximately” means ±10% of the value beingmodified.

In some embodiments, the amount of brigatinib in the disclosedpharmaceutical compositions is equal to or less than about 10 g, about9.5 g, about 9.0 g, about 8.5 g, about 8.0 g, about 7.5 g, about 7.0 g,about 6.5 g, about 6.0 g, about 5.5 g, about 5.0 g, about 4.5 g, about4.0 g, about 3.5 g, about 3.0 g, about 2.5 g, about 2.0 g, about 1.5 g,about 1.0 g, about 0.95 g, about 0.9 g, about 0.85 g, about 0.8 g, about0.75 g, about 0.7 g, about 0.65 g, about 0.6 g, about 0.55 g, about 0.5g, about 0.45 g, about 0.4 g, about 0.35 g, about 0.3 g, about 0.25 g,about 0.2 g, about 0.15 g, about 0.1 g, about 0.09 g, about 0.08 g,about 0.07 g, about 0.06 g, about 0.05 g, about 0.04 g, about 0.03 g,about 0.02 g, about 0.01 g, about 0.009 g, about 0.008 g, about 0.007 g,about 0.006 g, about 0.005 g, about 0.004 g, about 0.003 g, about 0.002g, about 0.001 g, about 0.0009 g, about 0.0008 g, about 0.0007 g, about0.0006 g, about 0.0005 g, about 0.0004 g, about 0.0003 g, about 0.0002g, or about 0.0001 g. In some embodiments, the amount of one or more ofthe compounds as disclosed herein can be more than about 0.0001 g, about0.0002 g, about 0.0003 g, about 0.0004 g, about 0.0005 g, about 0.0006g, about 0.0007 g, about 0.0008 g, about 0.0009 g, about 0.001 g, about0.0015 g, about 0.002 g, about 0.0025 g, about 0.003 g, about 0.0035 g.about 0.004 g, about 0.0045 g, about 0.005 g, about 0.0055 g, about0.006 g, about 0.0065 g, about 0.007 g, about 0.0075 g, about 0.008 g,about 0.0085 g, about 0.009 g, about 0.0095 g, about 0.01 g, about 0.015g, about 0.02 g, about 0.025 g, about 0.03 g, about 0.035 g, about 0.04g, about 0.045 g, about 0.05 g, about 0.055 g, about 0.06 g, about 0.065g, about 0.07 g, about 0.075 g, about 0.08 g, about 0.085 g, about 0.09g, about 0.095 g, about 0.1 g, about 0.15 g, about 0.2 g, about 0.25 g,about 0.3 g, about 0.35 g, about 0.4 g, about 0.45 g, about 0.5 g, about0.55 g, about 0.6 g, about 0.65 g, about 0.7 g, about 0.75 g, about 0.8g, about 0.85 g, about 0.9 g, about 0.95 g, about 1 g, about 1.5 g,about 2 g, about 2.5, about 3 g, about 3.5, about 4 g, about 4.5 g,about 5 g, about 5.5 g, about 6 g, about 6.5 g, about 7 g, about 7.5 g,about 8 g, about 8.5 g, about 9 g, about 9.5 g, or about 10 g. As usedherein, “about” means ±10% of the value being modified.

In some embodiments, the amount of brigatinib in the disclosedpharmaceutical compositions ranges from about 0.0001 to about 10 g,about 0.0005 g to about 9 g, about 0.001 g to about 0.5 g, about 0.001 gto about 2 g, about 0.001 g to about 8 g, about 0.005 g to about 2 g,about 0.005 g to about 7 g, about 0.01 g to about 6 g, about 0.05 g toabout 5 g, about 0.1 g to about 4 g, about 0.5 g to about 4 g, or about1 g to about 3 g. As used herein, “about” means ±10% of the value beingmodified.

In some embodiments, the present disclosure provides pharmaceuticalcompositions for oral administration comprising at least one compound asdisclosed herein and at least one pharmaceutically acceptable excipientsuitable for oral administration. In some embodiments, the presentdisclosure provides pharmaceutical compositions for oral administrationcomprising: (i) a therapeutically effective amount of at least onecompound as disclosed herein; optionally (ii) an effective amount of atleast one second agent; and (iii) aat least one pharmaceuticallyacceptable excipient suitable for oral administration. In someembodiments, the pharmaceutical composition further comprises (iv) aneffective amount of at least one third agent.

In some embodiments, the pharmaceutical composition can be a liquidpharmaceutical composition suitable for oral consumption. Pharmaceuticalcompositions suitable for oral administration can be presented, forexample, as discrete dosage forms, such as capsules, cachets, ortablets, or liquids or aerosol sprays each containing a predeterminedamount of an active ingredient as a powder or in granules, a solution,or a suspension in an aqueous or non-aqueous liquid, an oil-in-wateremulsion, or a water-in-oil liquid emulsion. Such dosage forms can beprepared by any of the methods of pharmacy, but all methods include thestep of bringing the active ingredient into association with thecarrier, which constitutes one or more ingredients. In general, thepharmaceutical compositions are prepared by uniformly and intimatelyadmixing the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product intothe desired presentation. For example, a tablet can be prepared bycompression or molding, optionally with one or more accessoryingredients. Compressed tablets can be prepared by compressing in asuitable machine the active ingredient in a free-flowing form such aspowder or granules, optionally mixed with an excipient such as, but notlimited to, a binder, a lubricant, an inert diluent, and/or a surfaceactive or dispersing agent. Molded tablets can be made by molding in asuitable machine a mixture of the powdered compound moistened with aninert liquid diluent.

The tablets can be uncoated or coated by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearate canbe employed. Formulations for oral use can also be presented as hardgelatin capsules wherein the active ingredient can be mixed with aninert solid diluent, for example, calcium carbonate, calcium phosphateor kaolin, or as soft gelatin capsules wherein the active ingredient canbe mixed with water or an oil medium, for example, peanut oil, liquidparaffin or olive oil.

The present disclosure further encompasses in some embodiments anhydrouspharmaceutical compositions and dosage forms comprising at least oneactive ingredient. Water can facilitate the degradation of somecompounds. For example, water can be added (e.g., about 5%) in thepharmaceutical arts as a means of simulating long-term storage in orderto determine characteristics such as shelf-life or the stability offormulations over time. Anhydrous pharmaceutical compositions and dosageforms can be prepared using anhydrous or low moisture containingingredients and low moisture or low humidity conditions. For example,pharmaceutical compositions and dosage forms which contain lactose canbe made anhydrous if substantial contact with moisture and/or humidityduring manufacturing, packaging, and/or storage is expected. Ananhydrous pharmaceutical composition can be prepared and stored suchthat its anhydrous nature is maintained. Accordingly, anhydrouspharmaceutical compositions can be packaged using materials known toprevent exposure to water such that they can be included in suitableformulary kits. Examples of suitable packaging include, but are notlimited to, hermetically sealed foils, plastic or the like, unit dosecontainers, blister packs, and strip packs.

An active ingredient can be combined in an intimate admixture with apharmaceutical carrier according to conventional pharmaceuticalcompounding techniques. The carrier can take a wide variety of formsdepending on the form of preparation desired for administration. Inpreparing the pharmaceutical compositions for an oral dosage form, anyof the usual pharmaceutical media can be employed as carriers, such as,for example, water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents, and the like in the case of oral liquidpreparations (such as suspensions, solutions, and elixirs) or aerosols;or carriers such as starches, sugars, micro-crystalline cellulose,diluents, granulating agents, lubricants, binders, and disintegratingagents can be used in the case of oral solid preparations, in someembodiments without employing the use of lactose. In some embodiments,compounds can be admixed with lactose, sucrose, starch powder, celluloseesters of alkanoic acids, cellulose alkyl esters, talc, stearic acid,magnesium stearate, magnesium oxide, sodium and calcium salts ofphosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate,polyvinylpyrrolidone, and/or polyvinyl alcohol for subsequentformulation. For example, suitable carriers include powders, capsules,and tablets, with the solid oral preparations. In some embodiments,tablets can be coated by standard aqueous or nonaqueous techniques.

Non-limiting examples of binders suitable for use in pharmaceuticalcompositions and dosage forms disclosed herein include, but are notlimited to, corn starch, potato starch, and other starches, gelatin,natural and synthetic gums such as acacia, sodium alginate, alginicacid, other alginates, powdered tragacanth, guar gum, cellulose and itsderivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethylcellulose calcium, sodium carboxymethyl cellulose), polyvinylpyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropylmethyl cellulose, microcrystalline cellulose, and mixtures thereof.

Non-limiting examples of fillers suitable for use in the pharmaceuticalcompositions and dosage forms disclosed herein include, but are notlimited to, talc, calcium carbonate (e.g., granules or powder),microcrystalline cellulose, powdered cellulose, dextrates, kaolin,mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, andmixtures thereof.

Disintegrants can be used in the pharmaceutical compositions and dosageforms disclosed herein to provide tablets that disintegrate when exposedto an aqueous environment. Too much of a disintegrant can producetablets which can disintegrate in the bottle. Too little can beinsufficient for disintegration to occur and can thus alter the rate andextent of release of the active ingredient(s) from the dosage form.Thus, a sufficient amount of disintegrant that is neither too little nortoo much to detrimentally alter the release of the active ingredient(s)can be used to prepare the pharmaceutical compositions and the dosageforms disclosed herein. The amount of disintegrant can vary based uponthe type of formulation and mode of administration, and can be readilydiscernible to those of ordinary skill in the art. For example, in someembodiments, about 0.5 to about 15 total weight percent of at least onedisintegrant may be used. In some embodiments, about 1 to about 5 totalweight percent of at least disintegrant can be used in thepharmaceutical composition. Disintegrants that can be used include, butare not limited to, agar agar, alginic acid, calcium carbonate,microcrystalline cellulose, croscarmellose sodium, crospovidone,polacrilin potassium, sodium starch glycolate, potato or tapioca starch,other starches, pre-gelatinized starch, other starches, clays, otheralgins, other celluloses, gums, and mixtures thereof.

Lubricants which can be used in pharmaceutical compositions and dosageforms disclosed herein include, but are not limited to, calciumstearate, magnesium stearate, mineral oil, light mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethylaureate, agar, syloidsilica gel, coagulated aerosol of synthetic silica, and mixturesthereof. A lubricant can optionally be added in an amount of less thanabout 1 total weight percent of the pharmaceutical composition.

When aqueous suspensions and/or elixirs are chosen for oraladministration, the pharmaceutical compositions may further comprise atleast one additional agent chosen from sweetening agents, flavoringagents, coloring matters, dyes, emulsifying agents, suspending agents,and diluents (e.g., water, ethanol, propylene glycol, glycerin and thelike).

Surfactants which can be included in the pharmaceutical compositions anddosage forms disclosed herein include, but are not limited to,hydrophilic surfactants, lipophilic surfactants, and mixtures thereof.That is, a mixture of hydrophilic surfactants can be employed, a mixtureof lipophilic surfactants can be employed, or a mixture of at least onehydrophilic surfactant and at least one lipophilic surfactant can beemployed.

In some embodiments, hydrophilic surfactant(s) has an HLB value of atleast about 10, while lipophilic surfactant(s) has an HLB value of orless than about 10. An empirical parameter used to characterize therelative hydrophilicity and hydrophobicity of non-ionic amphiphiliccompounds is the hydrophilic-lipophilic balance (“HLB” value).Surfactants with lower HLB values are more lipophilic or hydrophobic,and have greater solubility in oils, while surfactants with higher HLBvalues are more hydrophilic, and have greater solubility in aqueoussolutions. Hydrophilic surfactants are generally considered to be thosecompounds having an HLB value greater than about 10, as well as anionic,cationic, or zwitterionic compounds for which the HLB scale is notgenerally applicable. Similarly, lipophilic (i.e., hydrophobic)surfactants are compounds having an HLB value equal to or less thanabout 10. However, HLB value of a surfactant is merely a rough guidegenerally used to enable formulation of industrial, pharmaceutical andcosmetic emulsions.

Hydrophilic surfactants can be either ionic or nonionic. Suitable ionicsurfactants include, but are not limited to, alkylammonium salts;fusidic acid salts; fatty acid derivatives of amino acids,oligopeptides, and polypeptides; glyceride derivatives of amino acids,oligopeptides, and polypeptides; lecithins and hydrogenated lecithins;lysolecithins and hydrogenated lysolecithins; phospholipids andderivatives thereof; lysophospholipids and derivatives thereof;carnitine fatty acid ester salts; salts of alkylsulfates; fatty acidsalts; sodium docusate; acylactylates; mono- and di-acetylated tartaricacid esters of mono- and di-glycerides; succinylated mono- anddi-glycerides; citric acid esters of mono- and di-glycerides; andmixtures thereof.

Within the aforementioned group, ionic surfactants include, but are notlimited to, lecithins, lysolecithin, phospholipids, lysophospholipidsand derivatives thereof; carnitine fatty acid ester salts; salts ofalkylsulfates; fatty acid salts; sodium docusate; acylactylates; mono-and di-acetylated tartaric acid esters of mono- and di-glycerides;succinylated mono- and di-glycerides; citric acid esters of mono- anddi-glycerides; and mixtures thereof.

Other non-limiting examples of ionic surfactants include ionized formsof lecithin, lysolecithin, phosphatidylcholine,phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid,phosphatidylserine, lysophosphatidylcholine,lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidicacid, lysophosphatidylserine, PEG-phosphatidylethanolamine,PVP-phosphatidylethanolamine, lactylic esters of fatty acids,stearoyl-2-1actylate, stearoyl lactylate, succinylated monoglycerides,mono/diacetylated tartaric acid esters of mono/diglycerides, citric acidesters of mono/diglycerides, cholylsarcosine, caproate, caprylate,caprate, laurate, myristate, palmitate, oleate, ricinoleate, linoleate,linolenate, stearate, lauryl sulfate, teracecyl sulfate, docusate,lauroyl carnitines, palmitoyl carnitines, myristoyl carnitines, andsalts and mixtures thereof.

No-limiting examples of hydrophilic non-ionic surfactants includealkylglucosides; alkylmaltosides; alkylthioglucosides; laurylmacrogolglycerides; polyoxyalkylene alkyl ethers such as polyethyleneglycol alkyl ethers; polyoxyalkylene alkylphenols such as polyethyleneglycol alkyl phenols; polyoxyalkylene alkyl phenol fatty acid esterssuch as polyethylene glycol fatty acids monoesters and polyethyleneglycol fatty acids diesters; polyethylene glycol glycerol fatty acidesters; polyglycerol fatty acid esters; polyoxyalkylene sorbitan fattyacid esters such as polyethylene glycol sorbitan fatty acid esters;hydrophilic transesterification products of a polyol with at least onemember of glycerides, vegetable oils, hydrogenated vegetable oils, fattyacids, and sterols; polyoxyethylene sterols, derivatives, and analoguesthereof; polyoxyethylated vitamins and derivatives thereof;polyoxyethylene-polyoxypropylene block copolymers; and mixtures thereof;polyethylene glycol sorbitan fatty acid esters and hydrophilictransesterification products of a polyol with at least one member oftriglycerides, vegetable oils, and hydrogenated vegetable oils. Thepolyol can be glycerol, ethylene glycol, polyethylene glycol, sorbitol,propylene glycol, pentaerythritol, or a saccharide.

Other hydrophilic-non-ionic surfactants include, but are not limited to,PEG-10 laurate, PEG-12 laurate, PEG-20 laurate, PEG-32 laurate, PEG-32dilaurate, PEG-12 oleate, PEG-15 oleate. PEG-20 oleate, PEG-20 dioleate,PEG-32 oleate, PEG-200 oleate, PEG-400 oleate, PEG-15 stearate, PEG-32distearate, PEG-40 stearate, PEG-100 stearate, PEG-20 dilaurate, PEG-25glyceryl trioleate, PEG-32 dioleate, PEG-20 glyceryl laurate, PEG-30glyceryl laurate, PEG-20 glyceryl stearate, PEG-20 glyceryl oleate,PEG-30 glyceryl oleate, PEG-30 glyceryl laurate, PEG-40 glyceryllaurate, PEG-40 palm kernel oil, PEG-50 hydrogenated castor oil, PEG-40castor oil, PEG-35 castor oil, PEG-60 castor oil, PEG-40 hydrogenatedcastor oil, PEG-60 hydrogenated castor oil, PEG-60 corn oil, PEG-6caprate/caprylate glycerides, PEG-8 caprate/caprylate glycerides,polyglyceryl-10 laurate, PEG-30 cholesterol, PEG-25 phyto sterol, PEG-30soya sterol, PEG-20 trioleate, PEG-40 sorbitan oleate, PEG-80 sorbitanlaurate, polysorbate 20, polysorbate 80, POE-9 lauryl ether, POE-23lauryl ether, POE-10 oleyl ether, POE-20 oleyl ether, POE-20 stearylether, tocopheryl PEG-100 succinate, PEG-24 cholesterol,polyglyceryl-10oleate, Tween 40, Tween 60, sucrose monostearate, sucrosemonolaurate, sucrose monopalmitate, PEG 10-100 nonyl phenol series, PEG15-100 octyl phenol series, and poloxamers.

Suitable lipophilic surfactants include, but are not limited to, fattyalcohols; glycerol fatty acid esters; acetylated glycerol fatty acidesters; lower alcohol fatty acids esters; propylene glycol fatty acidesters; sorbitan fatty acid esters; polyethylene glycol sorbitan fattyacid esters; sterols and sterol derivatives; polyoxyethylated sterolsand sterol derivatives; polyethylene glycol alkyl ethers; sugar esters;sugar ethers; lactic acid derivatives of mono- and di-glycerides;hydrophobic transesterification products of a polyol with at least onemember of glycerides, vegetable oils, hydrogenated vegetable oils, fattyacids and sterols; oil-soluble vitamins/vitamin derivatives; andmixtures thereof. Within this group, non-limiting examples of lipophilicsurfactants include glycerol fatty acid esters, propylene glycol fattyacid esters, and mixtures thereof, or are hydrophobictransesterification products of a polyol with at least one member ofvegetable oils, hydrogenated vegetable oils, and triglycerides.

In some embodiments, the pharmaceutical compositions and dosage formsdisclosed herein can include at least one solubilizer to ensure goodsolubilization and/or dissolution of a compound as disclosed herein andto minimize precipitation of the compound. This may be useful forpharmaceutical compositions for normal use, e.g., pharmaceuticalcompositions for injection. A solubilizer can also be added to increasethe solubility of the hydrophilic drug and/or other components, such assurfactants, or to maintain the pharmaceutical composition as a stableor homogeneous solution or dispersion.

Examples of suitable solubilizers include, but are not limited to, thefollowing: alcohols and polyols, such as ethanol, isopropanol, butanol,benzyl alcohol, ethylene glycol, propylene glycol, butanediols andisomers thereof, glycerol, pentaerythritol, sorbitol, mannitol,transcutol, dimethyl isosorbide, polyethylene glycol, polypropyleneglycol, polyvinylalcohol, hydroxypropyl methylcellulose and othercellulose derivatives, cyclodextrins and cyclodextrin derivatives;ethers of polyethylene glycols having an average molecular weight ofabout 200 to about 6000, such as tetrahydrofurfuryl alcohol PEG ether(glycofurol) or methoxy PEG; amides and other nitrogen-containingcompounds such as 2-pyrrolidone, 2-piperidone, ε-caprolactam,N-alkylpyrrolidone, N-hydroxyalkylpyrrolidone, N-alkylpiperidone,N-alkylcaprolactam, dimethylacetamide and polyvinylpyrrolidone; esterssuch as ethyl propionate, tributylcitrate, acetyl triethylcitrate,acetyl tributyl citrate, triethylcitrate, ethyl oleate, ethyl caprylate,ethyl butyrate, triacetin, propylene glycol monoacetate, propyleneglycol diacetate, ε-caprolactone and isomers thereof, δ-valerolactoneand isomers thereof, β-butyrolactone and isomers thereof; and othersolubilizers known in the art, such as dimethyl acetamide, dimethylisosorbide, N-methylpyrrolidones, monooctanoin, diethylene glycolmonoethyl ether, and water.

Mixtures of solubilizers can also be used. Examples include, but are notlimited to, triacetin, triethylcitrate, ethyl oleate, ethyl caprylate,dimethylacetamide, N-methylpyrrolidone, N-hydxoxyethylpyrrolidone,polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydxoxypropylcyclodextrins, ethanol, polyethylene glycol 200-100, glycofurol,transcutol, propylene glycol, and dimethyl isosorbide. In someembodiments, solubilizers include sorbitol, glycerol, triacetin, ethylalcohol, PEG-400, glycofurol, and propylene glycol.

The amount of solubilizer that can be included can vary with thecomposition. The amount of a given solubilizer can be limited to abioacceptable amount, which can be readily determined by one of skill inthe art. In some circumstances, it can be advantageous to includeamounts of solubilizers far in excess of bioacceptable amounts, forexample to maximize the concentration of the drug, with excesssolubilizer removed prior to providing the pharmaceutical composition toa subject using conventional techniques, such as distillation orevaporation. Thus, if present, the solubilizer can be present in anamount of about 10%, about 25%, about 50%, about 100%, or up to about200% by weight based on the total weight of the composition. In someembodiments, solubilizer can be present in an amount of about 5%, about2%, about 1% or even less. In some embodiments, solubilizer can bepresent in an amount of about 1% to about 100%, such as from about 5% toabout 25% by weight.

The pharmaceutical composition can further comprise at least onepharmaceutically acceptable excipient. Such excipients include, but arenot limited to, detackifiers, anti-foaming agents, buffering agents,polymers, antioxidants, preservatives, chelating agents,viscomodulators, tonicifiers, flavorants, colorants, oils, odorants,opacifiers, suspending agents, binders, fillers, plasticizers,lubricants, and mixtures thereof.

Non-limiting examples of preservatives include antioxidants, chelatingagents, antimicrobial preservatives, antifungal preservatives, alcoholpreservatives, acidic preservatives, and other preservatives. Exemplaryantioxidants include, but are not limited to, alpha tocopherol, ascorbicacid, acorbyl palmitate, butylated hydroxyanisole, butylatedhydroxytoluene, monothioglycerol, potassium metabisulfite, propionicacid, propyl gallate, sodium ascorbate, sodium bisulfite, sodiummetabisulfite, and sodium sulfite. Non-limiting examples of chelatingagents include ethylenediaminetetraacetic acid (EDTA), citric acidmonohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaricacid, malic acid, phosphoric acid, sodium edetate, tartaric acid, andtrisodium edetate. Exemplary antimicrobial preservatives include, butare not limited to, benzalkonium chloride, benzethonium chloride, benzylalcohol, bronopol, cetrimide, cetylpyridinium chloride, chiorhexidine,chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol,glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethylalcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.Exemplary antifungal preservatives include, but are not limited to,butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoicacid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodiumbenzoate, sodium propionate, and sorbic acid. Exemplary alcoholpreservatives include, but are not limited to, ethanol, polyethyleneglycol, phenol, phenolic compounds, bisphenol, chlorobutanol,hydroxybenzoate, and phenylethyl alcohol. Exemplary acidic preservativesinclude, but are not limited to, vitamin A, vitamin C, vitamin E,betacarotene, citric acid, acetic acid, dehydroacetic acid, ascorbicacid, sorbic acid, and phytic acid. Other preservatives include, but arenot limited to, tocopherol, tocopherol acetate, deteroxime mesylate,cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluene(BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ethersulfate (SLES), sodium bisulfite, sodium metabisulfite, potassiumsulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben,Germall 115, Germaben II, Neolone, Kathon, and Euxyl. In certainembodiments, the preservative can be an anti-oxidant. In otherembodiments, the preservative can be a chelating agent.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukni nut, lavandin, lavender, lemon,Litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and combinations thereof.

In some embodiments, the composition disclosed herein may be oil/aqueousformulations. Oil/aqueous emulsion formulations can comprise at leastone emulsifier optionally with at least one fat and/oil. In someembodiments, at least one hydrophilic emulsifier can be included in thecompositions disclosed herein, optionally together with at least onelipophilic emulsifier, which may acts as a stabilizer. In someembodiments, both an oil and a fat can be used. The at least oneemulsifier optionally with at least one stabilizer may create at leastone emulsifying wax, which may form an emulsifying ointment base. Thisointment base may form an oily dispersed phase of cream formulations.Emulsifiers and emulsion stabilizers suitable for use in the disclosedformulations include, but are not limited to, Tween 60, Span 80,cetostearyl alcohol, myristyl alcohol, glyceryl monostearate, sodiumlauryl sulfate, glyceryl distearate alone or with a wax, and othermaterials well known in the art. In some cases, the solubility of theactive compound in the oil(s) likely to be used in the pharmaceuticalemulsion formulations can be low. Straight or branched chain, mono- ordibasic alkyl esters can aid solubility, such as di-isoadipate, isocetylstearate, propylene glycol diester of coconut fatty acids, isopropylmyristate, decyl oleate, isopropyl palmitate, butyl stearate,2-ethylhexyl palmitate or a blend of branched chain esters can be used.These can be used alone or in combination depending on the propertiesrequired. Alternatively, high melting point lipids such as white softparaffin and/or liquid paraffin or other mineral oils can be used.

In addition, an acid or a base can be incorporated into thepharmaceutical composition to facilitate processing, to enhancestability, or for other reasons. Examples of pharmaceutically acceptablebases include amino acids, amino acid esters, ammonium hydroxide,potassium hydroxide, sodium hydroxide, sodium hydrogen carbonate,aluminum hydroxide, calcium carbonate, magnesium hydroxide, magnesiumaluminum silicate, synthetic aluminum silicate, synthetic hydrocalcite,magnesium aluminum hydroxide, diisopropylethylamine, ethanolamine,ethylenediamine, triethanolamine, triethylamine, triisopropanolamine,trimethylamine, tris(hydroxymethyl)aminomethane (TRIS) and the like.Also suitable are bases that are salts of a pharmaceutically acceptableacid, such as acetic acid, acrylic acid, adipic acid, alginic acid,alkanesulfonic acid, amino acids, ascorbic acid, benzoic acid, boricacid, butyric acid, carbonic acid, citric acid, fatty acids, formicacid, fumaric acid, gluconic acid, hydroquinosulfonic acid, isoascorbicacid, lactic acid, maleic acid, oxalic acid, para-bromophenylsulfonicacid, propionic acid, p-toluenesulfonic acid, salicylic acid, stearicacid, succinic acid, tannic acid, tartaric acid, thioglycolic acid,toluenesulfonic acid, uric acid, and the like. Salts of polyproticacids, such as sodium phosphate, disodium hydrogen phosphate, and sodiumdihydrogen phosphate can also be used. When the base is a salt, thecation can be any convenient and pharmaceutically acceptable cation,such as ammonium, alkali metals, alkaline earth metals, and the like.Examples can include, but not limited to, sodium, potassium, lithium,magnesium, calcium and ammonium.

Non-limiting examples of suitable acids are pharmaceutically acceptableorganic or inorganic acids. Examples of suitable inorganic acidsinclude, but are not limited to, hydrochloric acid, hydrobromic acid,hydriodic acid, sulfuric acid, nitric acid, boric acid, phosphoric acid,and the like. Examples of suitable organic acids include, but are notlimited to, acetic acid, acrylic acid, adipic acid, alginic acid,alkanesulfonic acids, amino acids, ascorbic acid, benzoic acid, boricacid, butyric acid, carbonic acid, citric acid, fatty acids, formicacid, fumaric acid, gluconic acid, hydroquinosulfonic acid, isoascorbicacid, lactic acid, maleic acid, methanesulfonic acid, oxalic acid, parabromophenylsulfonic acid, propionic acid, p-toluenesulfonic acid,salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid,thioglycolic acid, toluenesulfonic acid, uric acid, and the like.

In some embodiments, provided herein are pharmaceutical compositions forparenteral administration containing at least one compound as disclosedherein and at least one pharmaceutically acceptable excipient suitablefor parenteral administration. In some embodiments, provided herein arepharmaceutical compositions for parenteral administration comprising:(i) an effective amount of at least one compound disclosed herein;optionally (ii) an effective amount of at least one second agent; and(iii) at least one pharmaceutically acceptable excipient suitable forparenteral administration. In some embodiments, the pharmaceuticalcomposition further comprises (iv) an effective amount of at least onethird agent.

The forms in which the disclosed pharmaceutical compositions can beincorporated for administration by injection include aqueous or oilsuspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, orpeanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueoussolution, and similar pharmaceutical vehicles. Aqueous solutions insaline are also conventionally used for injection. Ethanol, glycerol,propylene glycol, liquid polyethylene glycol, benzyl alcohol, and thelike (and suitable mixtures thereof), cyclodextrin derivatives, sodiumchloride, tragacanth gum, buffers, and vegetable oils can also beemployed.

Aqueous solutions in saline are also conventionally used for injection.Ethanol, glycerol, propylene glycol, liquid polyethylene glycol, and thelike (and suitable mixtures thereof), cyclodextrin derivatives, andvegetable oils can also be employed. The proper fluidity can bemaintained, for example, by the use of a coating, such as lecithin, forthe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like.

In some embodiments, the active ingredient can also be administered byinjection as a composition with suitable carriers including, but notlimited to, saline, dextrose, or water, or with cyclodextrin (e.g.,Captisol), cosolvent solubilization (e.g., propylene glycol) or micellarsolubilization (e.g., Tween 80).

Sterile injectable solutions can be prepared by incorporating a compoundas disclosed herein in the required amount in the appropriate solventwith various other ingredients as enumerated above, as appropriate,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the various sterilized active ingredients into asterile vehicle which contains the basic dispersion medium and theappropriate other ingredients from those enumerated above. In the caseof sterile powders for the preparation of sterile injectable solutions,certain methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional ingredient from a previously sterile-filtered solutionthereof.

The sterile injectable preparation can also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that can be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil can be employed,including synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use. Injectable compositions can contain from about 0.1%to about 5% w/w of a compound as disclosed herein.

In some embodiments, provided herein are pharmaceutical compositions fortopical (e.g., transdermal) administration comprising at least onecompound as disclosed herein and at least one pharmaceuticallyacceptable excipient suitable for topical administration. In someembodiments, provided herein are pharmaceutical compositions for topicaladministration comprising (i) an effective amount of at least onecompound disclosed herein; optionally (ii) an effective amount of atleast one second agent; and (iii) at least one pharmaceuticallyacceptable excipients suitable for topical administration. In someembodiments, the pharmaceutically acceptable composition furthercomprises (iv) an effective amount of at least one third agent.

Pharmaceutical compositions provided herein can be formulated intopreparations in solid, semi-solid, or liquid forms suitable for local ortopical administration, such as gels, water soluble jellies, linements,creams, lotions, suspensions, foams, powders, slurries, ointments,solutions, oils, pastes, suppositories, sprays, emulsions, salinesolutions, dimethylsulfoxide (DMSO)-based solutions. In general,carriers with higher densities are capable of providing an area with aprolonged exposure to the active ingredients. In contrast, a solutionformulation can provide more immediate exposure of the active ingredientto the chosen area. For example, an ointment formulation can have eithera paraffinic or a water-miscible base. Alternatively, the activeingredient can be formulated in a cream with an oil-in-water cream base.The aqueous phase of the cream base can include, for example at leastabout 30% w/w of a polyhydric alcohol such as propylene glycol,butane-1,3-diol, mannitol, sorbitol, glycerol, polyethylene glycol andmixtures thereof.

The pharmaceutical compositions also can comprise suitable solid or gelphase carriers or excipients, which are compounds that allow increasedpenetration of, or assist in the delivery of, therapeutic moleculesacross the stratum corneum permeability barrier of the skin. There aremany of these penetration-enhancing molecules known to those trained inthe art of topical formulation. Examples of such carriers and excipientsinclude, but are not limited to, humectants (e.g., urea), glycols (e.g.,propylene glycol), alcohols (e.g., ethanol), fatty acids (e.g., oleicacid), surfactants (e.g., isopropyl myristate and sodium laurylsulfate), pyrrolidones, glycerol monolaurate, sulfoxides, terpenes(e.g., menthol), amines, amides, alkanes, alkanols, water, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin, and polymers such as polyethylene glycols.

Another exemplary formulation for use in the disclosed methods employstransdermal delivery devices (“patches”). Such transdermal patches canbe used to provide continuous or discontinuous infusion of a compound asprovided herein in controlled amounts, either with or without anotheragent. Patches can be either of the reservoir and porous membrane typeor of a solid matrix variety. In either case, the active agent can bedelivered continuously from the reservoir or microcapsules through amembrane into the active agent permeable adhesive, which is in contactwith the skin or mucosa of the recipient. If the active agent isabsorbed through the skin, a controlled and predetermined flow of theactive agent can be administered to the recipient. In the case ofmicrocapsules, the encapsulating agent can also function as themembrane.

The construction and use of transdermal patches for the delivery ofpharmaceutical agents is well known in the art. See, e.g., U.S. Pat.Nos. 5,023,252, 4,992,445 and 5,001,139. Such patches can be constructedfor continuous, pulsatile, or on demand delivery of pharmaceuticalagents.

Suitable devices for use in delivering intradermal pharmaceuticallyacceptable compositions described herein include short needle devicessuch as those described in U.S. Pat. Nos. 4,886,499; 5,190,521;5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496; and 5,417,662.Intradermal compositions can be administered by devices which limit theeffective penetration length of a needle into the skin, such as thosedescribed in PCT publication WO 99/34850 and functional equivalentsthereof. Jet injection devices which deliver liquid vaccines to thedermis via a liquid jet injector and/or via a needle which pierces thestratum corneum and produces a jet which reaches the dermis aresuitable. Jet injection devices are described, for example, in U.S. Pat.Nos. 5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189;5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335;5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880;4,940,460; and PCT publications WO97/37705 and WO 97/13537. Ballisticpowder/particle delivery devices which use compressed gas to acceleratevaccine in powder form through the outer layers of the skin to thedermis are suitable. Alternatively or additionally, conventionalsyringes can be used in the classical mantoux method of intradermaladministration.

Topically-administrable formulations can, for example, comprise fromabout 1% to about 10% (w/w) of a disclosed compound, although theconcentration of the compound of Formula I can be as high as thesolubility limit of the compound in the solvent. In some embodiments,topically-administrable formulations can, for example, include fromabout 0.001% to about 10% (w/w) compound, about 1% to about 9% (w/w)compound, such as from about 1% to about 8% (w/w), further such as fromabout 1% to about 7% (w/w), further such as from about 1% to about 6%(w/w), further such as from about 1% to about 5% (w/w), further such asfrom about 1% to about 4% (w/w), further such as from about 1% to about3% (w/w), further such as from about 1% to about 2% (w/w), and furthersuch as from about 0.1% to about 1% (w/w) compound. In some embodiments,the topical formulation includes about 0.1 mg to about 150 mgadministered one to four, such as one or two times daily. Formulationsfor topical administration can further comprise one or more of theadditional pharmaceutically acceptable excipients described herein.

In some embodiments, provided herein are pharmaceutical compositions forinhalation administration comprising at least one compound as disclosedherein and at least one pharmaceutically acceptable excipients suitablefor topical administration. In some embodiments, provided herein arepharmaceutical compositions for inhalation administration comprising:(i) an effective amount of at least one compound disclosed herein;optionally (ii) an effective amount of at least one second agent; and(iii) at least one pharmaceutically acceptable excipient suitable forinhalation administration. In some embodiments, the pharmaceuticalcomposition further comprises: (iv) an effective amount of at least onethird agent.

Pharmaceutical compositions for inhalation or insufflation includesolutions and suspensions in pharmaceutically acceptable, aqueous ororganic solvents, or mixtures thereof and powders. The liquid or solidpharmaceutical compositions can contain suitable pharmaceuticallyacceptable excipients as described herein. For example, suitableexcipients include, but are not limited to, saline, benzyl alcohol andfluorocarbons. In some embodiments, the pharmaceutical compositions areadministered by the oral or nasal respiratory route for local orsystemic effect. Pharmaceutical compositions in pharmaceuticallyacceptable solvents can be nebulized by use of inert gases. Nebulizedsolutions can be inhaled directly from the nebulizing device or thenebulizing device can be attached to a face mask tent, or intermittentpositive pressure breathing machine. Solution, suspension, or powderpharmaceutical compositions can be administered, e.g., orally ornasally, from devices that deliver the formulation in an appropriatemanner.

In some embodiments, provided herein are pharmaceutical compositions forophthalmic administration comprising at least one compound as disclosedherein and at least one pharmaceutically acceptable excipient suitablefor ophthalmic administration. Pharmaceutical compositions suitable forocular administration can be presented as discrete dosage forms, such asdrops or sprays each containing a predetermined amount of an activeingredient, a solution, or a suspension in an aqueous or non-aqueousliquid, an oil-in-water emulsion, or a water-in-oil liquid emulsion.Other administration forms include intraocular injection, intravitrealinjection, topically, or through the use of a drug eluting device,microcapsule, implant, or microfluidic device. In some cases, thecompounds as disclosed herein are administered with a carrier orexcipient that increases the intraocular penetrance of the compound suchas an oil and water emulsion with colloid particles having an oily coresurrounded by an interfacial film. It is contemplated that all localroutes to the eye can be used including topical, subconjunctival,periocular, retrobulbar, subtenon, intracameral, intravitreal,intraocular, subretinal, juxtascleral and suprachoroidal administration.Systemic or parenteral administration can be feasible including, but notlimited to, intravenous, subcutaneous, and oral delivery. An exemplarymethod of administration can be intravitreal or subtenon injection ofsolutions or suspensions, or intravitreal or subtenon placement ofbioerodible or non-bioerodible devices, or by topical ocularadministration of solutions or suspensions, or posterior juxtascleraladministration of a gel or cream formulation.

Eye drops can be prepared by dissolving the active ingredient in asterile aqueous solution such as physiological saline, bufferingsolution, etc., or by combining powder compositions to be dissolvedbefore use. Other vehicles can be chosen, as is known in the art,including, but not limited to: balance salt solution, saline solution,water soluble polyethers such as polyethyene glycol, polyvinyls, such aspolyvinyl alcohol and povidone, cellulose derivatives such asmethylcellulose and hydroxypropyl methylcellulose, petroleum derivativessuch as mineral oil and white petrolatum, animal fats such as lanolin,polymers of acrylic acid such as carboxypolymethylene gel, vegetablefats such as peanut oil and polysaccharides such as dextrans, andglycosaminoglycans such as sodium hyaluronate. In some embodiments,additives ordinarily used in the eye drops can be added. Such additivesinclude isotonizing agents (e.g., sodium chloride, etc.), buffer agent(e.g., boric acid, sodium monohydrogen phosphate, sodium dihydrogenphosphate, etc.), preservatives (e.g., benzalkonium chloride,benzethonium chloride, chlorobutanol, etc.), thickeners (e.g.,saccharide such as lactose, mannitol, maltose, etc.; e.g., hyaluronicacid or its salt such as sodium hyaluronate, potassium hyaluronate,etc.; e.g., mucopolysaccharide such as chondritin sulfate, etc.; e.g.,sodium polyacrylate, carboxyvinyl polymer, crosslinked polyacrylate,polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose,hydroxypropyl methylcellulose, hydroxyethyl cellulose, carboxymethylcellulose, hydroxypropyl cellulose or other agents known to thoseskilled in the art).

In some cases, the colloid particles include at least one cationic agentand at least one non-ionic surfactant such as a poloxamer, tyloxapol, apolysorbate, a polyoxyethylene castor oil derivative, a sorbitan ester,or a polyoxyl stearate. In some cases, the cationic agent can beselected from an alkylamine, a tertiary alkyl amine, a quarternaryammonium compound, a cationiclipid, an amino alcohol, a biguanidinesalt, a cationic compound or a mixture thereof. In some cases, thecationic agent can be a biguanidine salt such as chlorhexidine,polyaminopropyl biguanidine, phenformin, alkylbiguanidine, or a mixturethereof. In some cases, the quaternary ammonium compound can be abenzalkonium halide, lauralkonium halide, cetrimide,hexadecyltrimethylammonium halide, tetradecyltrimethylammonium halide,dodecyltrimethylammonium halide, cetrimonium halide, benzethoniumhalide, behenalkonium halide, cetalkonium halide, cetethyldimoniumhalide, cetylpyridinium halide, benzododecinium halide, chiorallylmethenamine halide, rnyristylalkonium halide, stearalkonium halide or amixture of two or more thereof. In some cases, cationic agent can be abenzalkonium chloride, lauralkonium chloride, benzododecinium bromide,benzethenium chloride, hexadecyltrimethylammonium bromide,tetradecyltrimethylammonium bromide, dodecyltrimethylammonium bromide ora mixture of two or more thereof. In some cases, the oil phase can bemineral oil and light mineral oil, medium chain triglycerides (MCT),coconut oil; hydrogenated oils comprising hydrogenated cottonseed oil,hydrogenated palm oil, hydrogenate castor oil or hydrogenated soybeanoil; polyoxyethylene hydrogenated castor oil derivatives comprisingpoluoxyl-40 hydrogenated castor oil, polyoxyl-60 hydrogenated castor oilor polyoxyl-100 hydrogenated castor oil.

In some embodiments, the amount of a compound as disclosed herein in theformulation can be about 0.5% to about 20%, 0.5% to about 10%, or about1.5% w/w.

In some embodiments, provided herein are pharmaceutical compositions forcontrolled release administration comprising at least one compound asdisclosed herein and at least one pharmaceutically acceptable excipientsuitable for controlled release administration. In some embodiments,provided herein are pharmaceutical compositions for controlled releaseadministration comprising: (i) an effective amount of at least onecompound disclosed herein; optionally (ii) an effective amount of atleast one second agent; and (iii) at least one pharmaceuticallyacceptable excipient suitable for controlled release administration. Insome embodiments, the pharmaceutical composition further comprises: (iv)an effective amount of at least one third agent.

Active agents such as the compounds provided herein can be administeredby controlled release means or by delivery devices that are well knownto those of ordinary skill in the art. Examples include, but are notlimited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899;3,536,809; 3,598,123; and 4,008,719; 5,674,533; 5,059,595; 5,591,767;5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,639,480; 5,733,566;5,739,108; 5,891,474; 5,922,356; 5,972,891; 5,980,945; 5,993,855;6,045,830; 6,087,324; 6,113,943; 6,197,350; 6,248,363; 6,264,970;6,267,981; 6,376,461; 6,419,961; 6,589,548; 6,613,358; 6,699,500 each ofwhich is incorporated herein by reference. Such dosage forms can be usedto provide slow or controlled release of one or more active agentsusing, for example, hydropropylmethyl cellulose, other polymer matrices,gels, permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes, microspheres, or a combination thereof toprovide the desired release profile in varying proportions. Suitablecontrolled release formulations known to those of ordinary skill in theart, including those described herein, can be readily selected for usewith the active agents provided herein. Thus, the pharmaceuticalcompositions provided encompass single unit dosage forms suitable fororal administration such as, but not limited to, tablets, capsules,gelcaps, and caplets that are adapted for controlled release.

All controlled release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non controlledcounterparts. In some embodiments, the use of a controlled releasepreparation in medical treatment can be characterized by a minimum ofdrug substance being employed to cure or control the disease, disorder,or condition in a minimum amount of time. Advantages of controlledrelease formulations include extended activity of the drug, reduceddosage frequency, and increased subject compliance. In addition,controlled release formulations can be used to affect the time of onsetof action or other characteristics, such as blood levels of the drug,and can thus affect the occurrence of side (e.g., adverse) effects.

In some embodiments, controlled release formulations are designed toinitially release an amount of a compound as disclosed herein thatpromptly produces the desired therapeutic effect, and gradually andcontinually release other amounts of the compound to maintain this levelof therapeutic or prophylactic effect over an extended period of time.In order to maintain this constant level of the compound in the body,the compound should be released from the dosage form at a rate that willreplace the amount of drug being metabolized and excreted from the body.Controlled release of an active agent can be stimulated by variousconditions including, but not limited to, pH, temperature, enzymes,water, or other physiological conditions or compounds.

In certain embodiments, the pharmaceutical composition can beadministered using intravenous infusion, an implantable osmotic pump, atransdermal patch, liposomes, or other modes of administration. In someembodiments, a pump can be used (see, Sefton, CRC Crit. Ref Biomed. Eng.14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Sandek et al., N.Engl. J. Med. 321:574 (1989)). In another embodiment, polymericmaterials can be used. In yet another embodiment, a controlled releasesystem can be placed in a subject at an appropriate site determined by apractitioner of skill, i.e., thus requiring only a fraction of thesystemic dose (see, e.g., Goodson, Medical Applications of ControlledRelease, 115-138 (vol. 2, 1984). Other controlled release systems arediscussed in the review by Langer, Science 249:1527-1533 (1990). The atleast one active agent can be dispersed in a solid inner matrix, e.g.,polymethylmethacrylate, polybutylmethacrylate, plasticized orunplasticized polyvinylchloride, plasticized nylon, plasticizedpolyethyleneterephthalate, natural rubber, polyisoprene,polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetatecopolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonatecopolymers, hydrophilic polymers such as hydrogels of esters of acrylicand methacrylic acid, collagen, cross-linked polyvinylalcohol andcross-linked partially hydrolyzed polyvinyl acetate, that is surroundedby an outer polymeric membrane, e.g., polyethylene, polypropylene,ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers,ethylene/vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride,vinylchloride copolymers with vinyl acetate, vinylidene chloride,ethylene and propylene, ionomer polyethylene terephthalate, butyl rubberepichlorohydrin rubbers, ethylene/vinyl alcohol copolymer,ethylene/vinyl acetate/vinyl alcohol terpolymer, andethylene/vinyloxyethanol copolymer, that is insoluble in body fluids.The at least one active agent then diffuses through the outer polymericmembrane in a release rate controlling step. The percentage of at leastone active agent in such parenteral compositions can depend on thespecific nature thereof, as well as the needs of the subject.

A compound described herein can be delivered in the form ofpharmaceutically acceptable compositions which comprise atherapeutically effective amount of at least one compound disclosedherein and/or at least one additional therapeutic agent, such as achemotherapeutic, formulated together with at least one pharmaceuticallyacceptable excipient. In some embodiments, only a compound providedherein without an additional therapeutic agent can be included in thedosage form. In some instances, the compound described herein and theadditional therapeutic agent are administered in separate pharmaceuticalcompositions and can (e.g., because of different physical and/orchemical characteristics) be administered by different routes (e.g., onetherapeutic can be administered orally, while the other can beadministered intravenously). In other instances, the compound describedherein and the additional therapeutic agent can be administeredseparately, but via the same route (e.g., both orally or bothintravenously). In still other instances, the compound described hereinand the additional therapeutic agent can be administered in the samepharmaceutical composition.

The selected dosage level will depend upon a variety of factorsincluding, for example, the activity of the particular compoundemployed, the severity of the condition, the route of administration,the time of administration, the rate of excretion or metabolism of theparticular compound being employed, the rate and extent of absorption,the duration of the treatment, administration of other drugs, compoundsand/or materials used in combination with the particular compoundemployed, the age, sex, weight, condition, general health and priormedical history of the patient being treated, and like factors wellknown in the medical arts.

The dosage level can also be informed by in vitro or in vivo assayswhich can optionally be employed to help identify optimal dosage ranges.One guide to effective doses can be extrapolated from dose-responsecurves derived from in vitro or animal model test systems. Furthermore,after formulation with an appropriate pharmaceutically acceptablecarrier in a desired dosage, the compositions as disclosed herein can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bytransdermal patch, powders, ointments, or drops), sublingually, bucally,as an oral or nasal spray, or the like.

In general, a suitable daily dose of a compound described herein and/ora chemotherapeutic will be that amount of the compound which, in someembodiments, can be the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above. In some embodiments, the dose of the compoundsdescribed herein for a patient, when used for the indicated effects,will range from about 0.0001 mg to about 100 mg per day, or about 0.001mg to about 100 mg per day, or about 0.01 mg to about 100 mg per day, orabout 0.1 mg to about 100 mg per day, or about 0.1 mg to about 125 mgper day, or about 0.0001 mg to about 500 mg per day, or about 0.001 mgto about 500 mg per day, or about 0.01 mg to about 1000 mg per day, orabout 0.01 mg to about 500 mg per day, or about 0.1 mg to about 500 mgper day, or about 1 mg to about 25 mg per day, or about 1 mg to about 50mg per day, or about 5 mg to about 40 mg per day. An exemplary dosagecan be about 10 to about 30 mg per day. In some embodiments, for a 70 kghuman, a suitable dose would be about 0.05 to about 7 g/day, such asabout 0.05 to about 2 g/day. In some embodiments, the daily oral dose isabout 30 mg, about 90 mg, about 150 mg, or about 180 mg. As used herein,“about” means ±5% of the value being modified. Actual dosage levels ofthe active ingredients in the pharmaceutical compositions describedherein can be varied so as to obtain an amount of the active ingredientwhich is effective to achieve the desired therapeutic response for aparticular patient, composition, and mode of administration, withoutbeing toxic to the patient. In some instances, dosage levels below thelower limit of the aforesaid range can be more than adequate, while inother cases still larger doses can be employed without causing anyharmful side effect, e.g., by dividing such larger doses into severalsmall doses for administration throughout the day.

In some embodiments, the compounds can be administered daily, everyother day, three times a week, twice a week, weekly, bi-weekly, oranother intermittent schedule. The dosing schedule can include a “drugholiday,” i.e., the drug can be administered for two weeks on, one weekoff, or three weeks on, one week on, or four weeks on, one week off,etc., or continuously, without a drug holiday.

In some embodiments, a compound as provided herein can be administeredin multiple doses. Dosing can be about once, twice, three times, fourtimes, five times, six times, or more than six times per day. Dosing canbe about once a month, about once every two weeks, about once a week, orabout once every other day. In another embodiment, a compound asdisclosed herein and another agent are administered together about onceper day to about 6 times per day. For example, the compound can beadministered one or more times per day on a weekly basis (e.g., everyMonday) indefinitely or for a period of weeks, e.g., 4-10 weeks.Alternatively, it can be administered daily for a period of days (e.g.,2-10 days) followed by a period of days (e.g., 1-30 days) withoutadministration of the compound, with that cycle repeated indefinitely orfor a given number of repetitions, e.g., 4-10 cycles. As an example, acompound provided herein can be administered daily for 5 days, thendiscontinued for 9 days, then administered daily for another 5 dayperiod, then discontinued for 9 days, and so on, repeating the cycleindefinitely, or for a total of 4-10 times. In another embodiment, theadministration of a compound as provided herein and an agent continuesfor less than about 7 days. In yet another embodiment, theadministration continues for more than about 6, about 10, about 14,about 28 days, about two months, about six months, or about one year. Insome cases, continuous dosing can be achieved and maintained as long asnecessary.

Administration of the pharmaceutical compositions as disclosed hereincan continue as long as necessary. In some embodiments, an agent asdisclosed herein can be administered for more than about 1, about 2,about 3, about 4, about 5, about 6, about 7, about 14, or about 28 days.In some embodiments, an agent as disclosed herein can be administeredfor less than about 28, about 14, about 7, about 6, about 5, about 4,about 3, about 2, or about 1 day. In some embodiments, an agent asdisclosed herein can be administered chronically on an ongoing basis,e.g., for the treatment of chronic effects.

When administered for the treatment or inhibition of a particulardisease state or disorder, the effective dosage of the compound asdisclosed herein can vary depending upon the particular compoundutilized, the mode of administration, the condition, and severitythereof, of the condition being treated, as well as the various physicalfactors related to the individual being treated. In some embodiments,the effective systemic dose of the compound will typically be in therange of about 0.01 to about 500 mg of compound per kg of patient bodyweight, such as about 0.1 to about 125 mg/kg, and in some cases about 1to about 25 mg/kg, administered in single or multiple doses. Theprojected daily dosages are expected to vary with route ofadministration. Thus, parenteral dosing will often be at levels of about10% to about 20% of oral dosing levels. Generally, the compound can beadministered to patients in need of such treatment in a daily dose rangeof about 50 to about 2000 mg per patient. Administration can be once ormultiple times daily, weekly (or at some other multiple-day interval) oron an intermittent schedule.

In some embodiments, the dose of a compound as disclosed herein can beselected from 30, 60, 90, 120, 180, and 240 mg administered orally oncedaily. Another dosing regimen can include 90 mg administered orally oncedaily, or an oral 90 mg dose each day for 7 days followed by a 180 mgdose each day. In some embodiments, the compound being dosed isbrigatinib Form A.

Since the compounds described herein can be administered in combinationwith other treatments (such as additional chemotherapeutics, radiationor surgery), the doses of each agent or therapy can be lower than thecorresponding dose for single-agent therapy. The dose for single agenttherapy can range from, for example, about 0.0001 to about 200 mg, orabout 0.001 to about 100 mg, or about 0.01 to about 100 mg, or about 0.1to about 100 mg, or about 1 to about 50 mg per kilogram of body weightper day.

When a compound provided herein is administered in a pharmaceuticalcomposition that comprises one or more agents, and one or more of theagents has a shorter half-life than the compound provided herein, unitdose forms of the agent(s) and the compound provided herein can beadjusted accordingly.

In some embodiments, provided herein are kits. The kits can include acompound or pharmaceutical composition as described herein, in suitablepackaging, and written material that can include instructions for use,discussion of clinical studies, listing of side effects, and the like.Kits are well suited for the delivery of solid oral dosage forms such astablets or capsules. Such kits can also include information, such asscientific literature references, package insert materials, clinicaltrial results, and/or summaries of these and the like, which indicate orestablish the activities and/or advantages of the pharmaceuticalcomposition, and/or which describe dosing, administration, side effects,drug interactions, or other information useful to the health careprovider. Such information can be based on the results of variousstudies, for example, studies using experimental animals involving invivo models and studies based on human clinical trials.

In some embodiments, a memory aid can be provided with the kit, e.g., inthe form of numbers next to the tablets or capsules whereby the numberscorrespond with the days of the regimen which the tablets or capsules sospecified should be ingested. Another example of such a memory aid canbe a calendar printed on the card, e.g., as follows “First Week, Monday,Tuesday, . . . etc . . . . Second Week, Monday, Tuesday, . . . ” etc.Other variations of memory aids will be readily apparent. A “daily dose”can be a single tablet or capsule or several tablets or capsules to betaken on a given day.

The kit can further contain another agent. In some embodiments, thecompound as disclosed herein and the agent are provided as separatepharmaceutical compositions in separate containers within the kit. Insome embodiments, the compound as disclosed herein and the agent areprovided as a single pharmaceutical composition within a container inthe kit. Suitable packaging and additional articles for use (e.g.,measuring cup for liquid preparations, foil wrapping to minimizeexposure to air, and the like) are known in the art and can be includedin the kit. In other embodiments, kits can further comprise devices thatare used to administer the active agents. Examples of such devicesinclude, but are not limited to, syringes, drip bags, patches, andinhalers. Kits described herein can be provided, marketed and/orpromoted to health providers, including physicians, nurses, pharmacists,formulary officials, and the like. Kits can also, in some embodiments,be marketed directly to the consumer.

An example of such a kit is a so-called blister pack. Blister packs arewell known in the packaging industry and are being widely used for thepackaging of pharmaceutical unit dosage forms (tablets, capsules, andthe like). Blister packs generally consist of a sheet of relativelystiff material covered with a foil of a usually transparent plasticmaterial. During the packaging process, recesses are formed in theplastic foil. The recesses have the size and shape of the tablets orcapsules to be packed. Next, the tablets or capsules are placed in therecesses and the sheet of relatively stiff material is sealed againstthe plastic foil at the face of the foil which is opposite from thedirection in which the recesses were formed. As a result, the tablets orcapsules are sealed in the recesses between the plastic foil and thesheet. The strength of the sheet is such that the tablets or capsulescan be removed from the blister pack by manually applying pressure onthe recesses whereby an opening is formed in the sheet at the place ofthe recess. The tablet or capsule can then be removed via said opening.

Kits can further comprise pharmaceutically acceptable vehicles that canbe used to administer one or more active agents. For example, if anactive agent is provided in a solid form that must be reconstituted forparenteral administration, the kit can comprise a sealed container of asuitable vehicle in which the active agent can be dissolved to form aparticulate free sterile solution that is suitable for parenteraladministration. Examples of pharmaceutically acceptable vehiclesinclude, but are not limited to: Water for Injection USP; aqueousvehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water-miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

The present disclosure further encompasses anhydrous pharmaceuticalcompositions and dosage forms comprising an active ingredient, sincewater can facilitate the degradation of some compounds. For example,water can be added (e.g., about 5%) in the pharmaceutical arts as ameans of simulating long-term storage in order to determinecharacteristics such as shelf-life or the stability of formulations overtime. Anhydrous pharmaceutical compositions and dosage forms can beprepared using anhydrous or low moisture containing ingredients and lowmoisture or low humidity conditions. For example, pharmaceuticalcompositions and dosage forms which contain lactose can be madeanhydrous if substantial contact with moisture and/or humidity duringmanufacturing, packaging, and/or storage is expected. An anhydrouspharmaceutical composition can be prepared and stored such that itsanhydrous nature is maintained. Accordingly, anhydrous pharmaceuticalcompositions can be packaged using materials known to prevent exposureto water such that they can be included in suitable formulary kits.Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastic or the like, unit dose containers,blister packs, and strip packs.

IV. Therapeutic Methods

In some embodiments, pharmaceutical compositions comprising at least onecrystalline form of brigatinib can be used for treating cancer, by theadministration of a therapeutically effective amount of thepharmaceutical composition to the subject in need thereof. In someembodiments, the cancer is an ALK+-driven cancer. In some embodiments,the cancer is non-small cell lung cancer.

A “therapeutically effective amount” is that amount effective fordetectable killing or inhibition of the growth or spread of cancercells; the size or number of tumors; or other measure of the level,stage, progression or severity of the cancer. The exact amount requiredcan vary from subject to subject, depending on the species, age, andgeneral condition of the subject, the severity of the disease, theparticular anticancer agent, its mode of administration, combinationtreatment with other therapies, and the like.

Disclosed herein are compounds having biological properties which makethem of interest for treating or modulating disease in which kinases canbe involved, symptoms of such disease, or the effect of otherphysiological events mediated by kinases. For instance, a number ofcompounds as disclosed herein have been shown to inhibit tyrosine kinaseactivity of ALK, fak and c-met, among other tyrosine kinases which arebelieved to mediate the growth, development and/or metastasis of cancer.A number of compounds as disclosed herein have also been found topossess potent in vitro activity against cancer cell lines, includingamong others karpas 299 cells. Such compounds are thus of interest forthe treatment of cancers, including solid tumors as well as lymphomasand including cancers which are resistant to other therapies.

In some embodiments, the cancer is an ALK+-driven cancer. In someembodiments, the cancer is non-small cell lung cancer. In someembodiments, the cancer is ALK-positive NSCLC. In some embodiments, thecancer is locally advanced or metastatic ALK-positive NSCLC. In someembodiments, the cancer/patient has previously been treated withcrizotinib or another tyrosine kinase inhibitor. In some embodiments,the cancer/patient has not previously been treated with an ALKinhibitor.

Such cancers include, but are not limited to, cancers of the breast, nonsmall cell lung cancer (NSCLC), neural tumors such as glioblastomas andneuroblastomas; esophaegeal carcinomas, soft tissue cancers such asrhabdomyosarcomas, among others; various forms of lymphoma such as anon-Hodgkin's lymphoma (NHL) known as anaplastic large-cell lymphoma(ALCL), various forms of leukemia; and including cancers which are ALKor c-met mediated.

Anaplastic Lymphoma Kinase (ALK) is a cell membrane-spannning receptortyrosine kinase, which belong to the insulin receptor subfamily. ALKreceptor tyrosine kinase (RTK) was initially identified due to itsinvolvement in the human non-Hodgkin lymphoma subtype known asanaplastic large-cell lymphoma (ALCL). ALK normally has a restricteddistribution in mammalian cells, being found at significant levels onlyin nervous system during embryonic development, suggesting a possiblerole for ALK in brain development (Duyster, J. Et al., Oncogene, 2001,20, 5623-5637).

In addition to its role in normal development, expression of thefull-length normal ALK has also been detected in cell lines derived froma variety of tumors such as neuroblastomas, neuroectodermal tumors(Lamant L. Et al., Am. J. Pathol., 2000, 156, 1711-1721;Osajima-Hakomori Y., et al., Am. J. Pathol. 2005, 167, 213-222) andglioblastoma (Powers C. et al., J. Biol. Chem. 2002, 277, 14153-14158;Grzelinski M. et al., Int. J. Cancer, 2005, 117, 942-951; Mentlein, R.Et al., J. Neurochem., 2002, 83, 747-753) as well as breast cancer andmelanoma lines (Dirk W G. Et al., Int. J. Cancer, 2002, 100, 49-56).

In common with other RTKs, translocations affect the ALK gene, resultingin expression of oncogenic fusion kinases, the most common of which isNPM-ALK. For example, approximately sixty percent of anaplastic largecell lymphomas (ALCL) are associated with a chromosome mutation thatgenerates a fusion protein consisting of nucleophosmin (NMP) and theintracellular domain of ALK. (Armitage, J. O. et al., Cancer: principleand practice of oncology, 6^(th) Edition, 2001, 2256-2316; kutok, J. L.& Aster J. C., J. Clin. Oncol., 2002, 20, 3691-3702; Wan, W. et al.,Blood, 2006, 107, 1617-1623. This mutant protein, NPM-ALK, possesses aconstitutively active tyrosine kinase domain that is responsible for itsoncogenic property through activation of downstream effectors (Falini, Band al., Blood, 1999, 94, 3509-3515; Morris, S. W. et al., Brit. J.Haematol., 2001, 113, 275-295). Experimental data have demonstrated thatthe aberrant expression of constitutively active ALK is directlyimplicated in the pathogenesis of ALCL and that inhibition of ALK canmarkedly impair the growth of ALK positive lymphoma cells (Kuefer, Mu etal., Blood, 1997, 90, 2901-2910; Bai, R. Y. et al., Exp. Hematol., 2001,29, 1082-1090; Slupianek, A. et al., Cancer Res., 2001, 61, 2194-2199;Turturro, F. et al., Clin. Cancer. Res., 2002, 8, 240-245). Theconstitutively activated chimeric ALK has also been demonstrated inabout 60% of inflammatory myofibroblastic tumors (IMTs), a slow growingsarcoma that mainly affects children and young adults (Lawrence, B. etal., Am. J. Pathol., 2000, 157, 377-384). Furthermore, recent reportshave also described the occurrence of a variant ALK fusion, TPM4-ALK, incases of squamous cell carcinoma (SCC) of the esophagus (Jazzi fr., etal., World J. Gastroenterol., 2006, 12, 7104-7112; Du X., et al., J.Mol. Med., 2007, 85, 863-875; Aklilu M., Semin. Radiat. Oncol., 2007,17, 62-69). Thus, ALK is one of the few examples of an RTK implicated inoncogenesis in both non-hematopoietic and hematopoietic malignancies.More recently, it has been shown that a small inversion withinchromosome 2p results in the formation of a fusion gene comprisinigportions of the echinoderm microtubule-associated protein-like 4 (EML4)gene and the anaplastic lymphoma kinase (ALK) gene in non-small-celllung cancer (NSCLC) cells (Soda M., et al., Nature, 2007, 448, 561-567).

In some embodiments, an ALK inhibitor can create durable cures when usedas a single therapeutic agent or combined with current chemotherapy forALCL, IMT, proliferative disorders, glioblastoma and other possiblesolid tumors cited herein, or, as a single therapeutic agent, could beused in a maintenance role to prevent recurrence in patients in need ofsuch a treatment.

Compounds as disclosed herein can be administered as part of a treatmentregimen in which the compound is the sole active pharmaceutical agent,or used in combination with one or more other therapeutic agents as partof a combination therapy. When administered as one component of acombination therapy, the therapeutic agents being administered can beformulated as separate compositions that are administered at the sametime or sequentially at different times (e.g., within 72 hours, 48hours, or 24 hours of one another), or the therapeutic agents can beformulated together in a single pharmaceutical composition andadministered simultaneously.

Thus, the administration of brigatinib in a form disclosed herein can bein conjunction with at least one additional therapeutic agent known tothose skilled in the art in the prevention or treatment of cancer, suchas radiation therapy or cytostatic agents, cytotoxic agents, otheranti-cancer agents and other drugs to ameliorate symptoms of the canceror side effects of any of the drugs. Non-limiting examples additionaltherapeutic agents include agents suitable for immunotherapy (such as,for example, PD-1 and PDL-1 inhibitors), antiangiogenesis (such as, forexample, bevacizumab), and/or chemotherapy.

If formulated as a fixed dose, such combination products employcompounds as disclosed herein within the accepted dosage ranges.Compounds as disclosed herein can also be administered sequentially withother anticancer or cytotoxic agents when a combination formulation isinappropriate. Compounds as disclosed herein can be administered priorto, simultaneously with, or after administration of the other anticanceror cytotoxic agent.

Currently, standard treatment of primary tumors consists of surgicalexcision, when appropriate, followed by either radiation orchemotherapy, and typically administered intravenously (IV). The typicalchemotherapy regime consists of either DNA alkylating agents, DNAintercalating agents, CDK inhibitors, or microtubule poisons. Thechemotherapy doses used are just below the maximal tolerated dose andtherefore dose limiting toxicities typically include, nausea, vomiting,diarrhea, hair loss, neutropenia and the like.

There are large numbers of antineoplastic agents available in commercialuse, in clinical evaluation and in pre-clinical development, which wouldbe selected for treatment of cancer by combination drug chemotherapy.And there are several major categories of such antineoplastic agents,namely, antibiotic-type agents, alkylating agents, antimetaboliteagents, hormonal agents, immunological agents, interferon-type agentsand a category of miscellaneous agents.

A first family of antineoplastic agents which can be used in combinationwith compounds as disclosed herein includesantimetabolite-type/thymidilate synthase inhibitor antineoplasticagents. Suitable antimetabolite antineoplastic agents can be selectedfrom, but not limited to, 5-FU-fibrinogen, acanthifolic acid,aminothiadiazole, brequinar sodium, carmofur, CibaGeigy CGP-30694,cyclopentyl cytosine, cytarabine phosphate stearate, cytarabineconjugates, Lilly DATHF, Merrel Dow DDFC, dezaguanine, dideoxycytidine,dideoxyguanosine, didox, Yoshitomi DMDC, doxifluridine, Wellcome EHNA,Merck & Co., EX-015, fazarabine, floxuridine, fludarabine phosphate,5fluorouracil, N-(21-furanidyl) fluorouracil, Daiichi Seiyaku FO-152,isopropyl pyrrolizine, Lilly LY-188011, Lilly LY-264618, methobenzaprim,methotrexate, Wellcome MZPES, norspermidine, NCI NSC-127716, NCINSC-264880, NCI NSC-39661, NCI NSC-612567, Warner-Lambert PALA,pentostatin, piritrexim, plicamycin, Asahi Chemical PL-AC, TakedaTAC788, thioguanine, tiazofurin, Erbamont TIF, trimetrexate, tyrosinekinase inhibitors, Taiho UFT and uricytin.

A second family of antineoplastic agents which can be used incombination with compounds as disclosed herein consists ofalkylating-type antineoplastic agents. Suitable alkylating-typeantineoplastic agents can be selected from, but not limited to, Shionogi254-S, aldo-phosphamide analogues, altretamine, anaxirone, BoehringerMannheim BBR-2207, bestrabucil, budotitane, Wakunaga CA-102,carboplatin, carmustine, Chinoin-139, Chinoin-153, chlorambucil,cisplatin, cyclophosphamide, American Cyanamid CL-286558, Sanofi CY-233,cyplatate, Degussa D 384, Sumimoto DACHP(Myr)2, diphenylspiromustine,diplatinum cytostatic, Erba distamycin derivatives, Chugai DWA-2114R,ITI E09, elmustine, Erbamont FCE-24517, estramustine phosphate sodium,fotemustine, Unimed G M, Chinoin GYKI-17230, hepsulfam, ifosfamide,iproplatin, lomustine, mafosfamide, mitolactolf Nippon Kayaku NK-121,NCI NSC-264395, NCI NSC-342215, oxaliplatin, Upjohn PCNU, prednimustine,Proter PTT-119, ranimustine, semustine, SmithKline SK&F-101772, YakultHonsha SN-22, spiromus-tine, Tanabe Seiyaku TA-077, tauromustine,temozolomide, teroxirone, tetraplatin and trimelamol.

A third family of antineoplastic agents which can be used in combinationwith compounds as disclosed herein includes antibiotic-typeantineoplastic agents. Suitable antibiotic-type antineoplastic agentscan be selected from, but not limited to, Taiho 4181-A, aclarubicin,actinomycin D, actinoplanone, Erbamont ADR-456, aeroplysinin derivative,Ajinomoto AN II, Ajinomoto AN3, Nippon Soda anisomycins, anthracycline,azino-mycin-A, bisucaberin, Bristol-Myers BL-6859, Bristol-MyersBMY-25067, Bristol-Myers BNY-25551, Bristol-Myers BNY-26605IBristolMyers BNY-27557, Bristol-Myers BMY-28438, bleomycin sulfate,bryostatin-1, Taiho C-1027, calichemycin, chromoximycin, dactinomycin,daunorubicin, Kyowa Hakko DC-102, Kyowa Hakko DC-79, Kyowa Hakko DC-88A,Kyowa Hakko, DC89-AI, Kyowa Hakko DC92-B, ditrisarubicin B, ShionogiDOB-41, doxorubicin, doxorubicin-fibrinogen, elsamicin-A, epirubicin,erbstatin, esorubicin, esperamicin-AI, esperamicin-Alb, ErbamontFCE21954, Fujisawa FK-973, fostriecin, Fujisawa FR-900482, glidobactin,gregatin-A, grincamycin, herbimycin, idarubicin, illudins, kazusamycin,kesarirhodins, Kyowa Hakko KM-5539, Kirin Brewery KRN-8602, Kyowa HakkoKT-5432, Kyowa Hakko KT-5594, Kyowa Hakko KT-6149, American CyanamidLL-D49194, Meiji Seika ME 2303, menogaril, mitomycin, mitoxantrone,SmithKline M-TAG, neoenactin, Nippon Kayaku NK-313, Nippon KayakuNKT-01, SRI International NSC-357704, oxalysine, oxaunomycin,peplomycin, pilatin, pirarubicin, porothramycin, pyrindanycin A, TobishiRA-I, rapamycin, rhizoxin, rodorubicin, sibanomicin, siwenmycin,Sumitomo SM5887, Snow Brand SN-706, Snow Brand SN-07, sorangicin-A,sparsomycin, SS Pharmaceutical SS-21020, SS Pharmaceutical SS-7313B, SSPharmaceutical SS-9816B, steffimycin B, Taiho 4181-2, talisomycin,Takeda TAN-868A, terpentecin, thrazine, tricrozarin A, Upjohn U-73975,Kyowa Hakko UCN-10028A, Fujisawa WF-3405, Yoshitomi Y-25024 andzorubicin.

A fourth family of antineoplastic agents which can be used incombination with compounds as disclosed herein includes a miscellaneousfamily of antineoplastic agents, such as tubulin interacting agents,topoisomerase II inhibitors, topoisomerase I inhibitors and hormonalagents, selected from, but not limited to, xcarotene,X-difluoromethyl-arginine, acitretin, Biotec AD-5, Kyorin AHC-52,alstonine, amonafide, amphethinile, amsacrine, Angiostat, ankinomycin,anti-neoplaston A10, antineoplaston A2, antineoplaston A3,antineoplaston A5, antineoplaston AS2-1F Henkel APD, aphidicolinglycinate, asparaginase, Avarol, baccharin, batracylin, benfluron,benzotript, Ipsen-Beaufour BIM-23015, bisantrene, BristoMyers BNY-40481,Vestar boron-10, bromofosfamide, Wellcome BW-502, Wellcome BW-773,caracemide, carmethizole hydrochloride, Ajinomoto CDAF,chlorsulfaquinoxalone, Chemes CHX-2053, Chemex CHX-100, Warner-LambertCI-921, WarnerLambert CI-937, Warner-Lambert CI-941, Warner-LambertC1958, clanfenur, claviridenone, ICN compound 1259, ICN compound 4711,Contracan, Yakult Honsha CPT-11, crisnatol, curaderm, cytochalasin B,cytarabine, cytocytin, Merz D-609, DABIS maleate, dacarbazine,datelliptinium, didemnin-B, dihaematoporphyrin ether, dihydrolenperone,dinaline, distamycin, Toyo Pharmar DM-341, Toyo Pharmar DM-75, DaiichiSeiyaku DN-9693, docetaxel elliprabin, elliptinium acetate, TsumuraEPMTC, the epothilones, ergotamine, etoposide, etretinate, fenretinide,Fujisawa FR-57704t gallium nitrate, genkwadaphnin, Chugai GLA-43, GlaxoGR-63178, grifolan NMF5N, hexadecylphosphocholine, Green Cross HO-221,homoharringtonine, hydroxyurea, BTG ICRF-187, ilmofosine, isoglutamine,isotretinoin, Otsuka JI-36, Ramot K-477, Otsuak K-76000Na, KurehaChemical K-AM, MECT Corp KI-8110, American Cyanamid L-623, leukoregulin,Ionidamine, Lundbeck LU 1121 Lilly LY-186641, NCI (US) MAP, marycin,Merrel Dow MDL-27048, Medco MEDR-340, merbarone, merocyanlnederivatives, methylanilinoacridine, Molecular Genetics MGI136,minactivin, mitonafide, mitoquidone mopidamol, motretinide, ZenyakuKogyo MST-16, N-(retinoyl)amino acids, Nisshin Flour Milling N-021,N-acylated-dehydroalanines, nafazatrom, Taisho NCU-190, nocodazolederivative, Normosang, NCI NSC-145813, NCI NSC-361456, NCI NSC-604782,NCI NSC-95580, ocreotide, Ono ONO-112, oquizanocine, Akzo Org-10172,paclitaxel, pancratistatin, pazelliptine, WarnerLambert PD-111707,Warner-Lambert PD-115934, Warner-Lambert PD-131141, Pierre FabrePE-1001, ICRT peptide D, piroxantrone, polyhaematoporphyrin, polypreicacid, Efamol porphyrin, probimane, procarbazine, proglumide, Invitronprotease nexin I, Tobishi RA-700, razoxane, Sapporo Breweries RBS,restrictin-P, retelliptine, retinoic acid, Rhone-Poulenc RP-49532,Rhone-Poulenc RP-56976, SmithKline SK&F-104864, Sumitomo SM-108, KuraraySMANCS, SeaPharm SP10094, spatol, spirocyclopropane derivatives,spirogermanium, Unimed, SS Pharmaceutical SS-554, strypoldinone,Stypoldione, Suntory SUN 0237, Suntory SUN 2071, superoxide dismutase,Toyama T-506, Toyama T-680, taxol, Teijin TEI-0303, teniposide,thaliblastine, Eastman Kodak TJB-29, tocotrienol, topotecan, Topostin,Teijin TT82, Kyowa Hakko UCN-01, Kyowa Hakko UCN-1028, ukrain, EastmanKodak USB-006, vinblastine sulfate, vincristine, vindesine,vinestramide, vinorelbine, vintriptol, vinzolidine, withanolides andYamanouchi YM.

Alternatively, the present compounds can also be used in co-therapieswith other anti-neoplastic agents, such as acemannan, aclarubicin,aldesleukin, alemtuzumab, alitretinoin, altretamine, amifostine,aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole,ANCER, ancestim, ARGLABIN, arsenic trioxide, BAM 002 (Novelos),bexarotene, bicalutamide, broxuridine, capecitabine, celmoleukin,cetrorelix, cladribine, clotrimazole, cytarabine ocfosfate, DA 3030(Dong-A), daclizumab, denileukin diftitox, deslorelin, dexrazoxane,dilazep, docetaxel, docosanol, doxercalciferol, doxifluridine,doxorubicin, bromocriptine, carmustine, cytarabine, fluorouracil, HITdiclofenac, interferon alfa, daunorubicin, doxorubicin, tretinoin,edelfosine, edrecolomab eflornithine, emitefur, epirubicin, epoetinbeta, etoposide phosphate, exemestane, exisulind, fadrozole, filgrastim,finasteride, fludarabine phosphate, formestane, fotemustine, galliumnitrate, gemcitabine, gemtuzumab zogamicin, gimeracil/oteracil/tegafurcombination, glycopine, goserelin, heptaplatin, human chorionicgonadotropin, human fetal alpha fetoprotein, ibandronic acid,idarubicin, (imiquimod, interferon alfa, interferon alfa, natural,interferon alfa-2, interferon alfa-2a, interferon alfa-2b, interferonalfa-NI, interferon alfa-n3, interferon alfacon1, interferon alpha,natural, interferon beta, interferon beta-Ia, interferon beta-Ib,interferon gamma, natural interferon gamma-Ia, interferon gamma-Ib,interleukin-I beta, iobenguane, irinotecan, irsogladine, Ianreotide, LC9018 (Yakult), leflunomide, lenograstim, lentinan sulfate, letrozole,leukocyte alpha interferon, leuprorelin, levamisole+fluorouracil,liarozole, lobaplatin, Ionidamine, lovastatin, masoprocol, melarsoprol,metoclopramide, mifepristone, miltefosine, mirimostim, mismatched doublestranded RNA, mitoguazone, mitolactol, mitoxantrone, molgramostim,nafarelin, naloxone+pentazocine, nartograstim, nedaplatin, nilutamide,noscapine, novel erythropoiesis stimulating protein, NSC 631570octreotide, oprelvekin, osaterone, oxaliplatin, paclitaxel, pamidronicacid, pegaspargase, peginterferon alfa-2b, pentosan polysulfate sodium,pentostatin, picibanil, pirarubicin, rabbit antithymocyte polyclonalantibody, polyethylene glycol interferon alfa-2a, porfimer sodium,raloxifene, raltitrexed, rasburicase, rhenium Re 186 etidronate, RIIretinamide, rituximab, romurtide, samarium (153 Sm) lexidronam,sargramostim, sizofiran, sobuzoxane, sonermin, strontium-89 chloride,suramin, tasonermin, tazarotene, tegafur, temoporfin, temozolomide,teniposide, tetrachlorodecaoxide, thalidomide, thymalfasin, thyrotropinalfa, topotecan, toremifene, tositumomab-iodine 131, trastuzumab,treosulfan, tretinoin, trilostane, trimetrexate, triptorelin, tumornecrosis factor alpha, natural, ubenimex, bladder cancer vaccine,Maruyama. vaccine, melanoma lysate vaccine, valrubicin, verteporfin,vinorelbine, VIRULIZIN, zinostatin stimalamer, or zoledronic acid;abarelix; AE 941 (Aeterna), ambamustine, antisense oligonucleotide,bcl-2 (Genta), APC 8015 (Dendreon), cetuximab, decitabine,dexaminoglutethimide, diaziquone, EL 532 (Elan), EM 800 (Endorecherche),eniluracil, etanidazole, fenretinidel filgrastim SDO1 (Amgen),fulvestrant, galocitabine, gastrin 17 immunogen, HLA-B7 gene therapy(Vical), granulocyte macrophage colony stimulating factor, histaminedihydrochloride, ibritumomab tiuxetan, ilomastat, IM 862 (Cytran),interleukin iproxifene, LDI 200 (Milkhaus), leridistim, lintuzumab, CA125 MAb (Biomira), cancer MAb (Japan Pharmaceutical Development), HER-2and Fc MAb (Medarex), idiotypic 105AD7 MAb (CRC Technology), idiotypicCEA MAb (Trilex), LYM iodine 131 MAb (Techniclone), polymorphicepithelial mucin-yttrium 90 MAb (Antisoma), marimastat, menogaril,mitumomab, motexafin, gadolinium, MX 6 (Galderma), nelarabine,nolatrexed, P 30 protein, pegvisomant, pemetrexed, porfiromycin,prinomastat, RL 0903 (Shire), rubitecan, satraplatin, sodiumphenylacetate, sparfosic acid, SRL 172 (SR Pharma), SU 5416 (SUGEN)y SU6668 (SUGEN), TA 077 (Tanabe), tetrathiomolybdate, thaliblastine,thrombopoietin, tin ethyl etiopurpurin, tirapazamine, cancer vaccine(Biomira), melanoma vaccine (New York University), melanoma vaccine(Sloan Kettering Institute), melanoma oncolysate vaccine (New YorkMedical College), viral melanoma cell lysates vaccine (Royal NewcastleHospital), or valspodar.

V. Synthesis of Brigatinib Form A

The following representative synthesis of brigatinib Form A containsadditional information, exemplification and guidance which can beadapted to the practice of the invention in its various embodiments andthe equivalents thereof.

Examples are intended to help illustrate the invention, and are notintended to, nor should they be construed to, limit its scope. Indeed,various modifications of the invention, and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art upon review of this document,including the examples which follow and the references to the scientificand patent literature cited herein.

The contents of those cited references are incorporated herein byreference to help illustrate the state of the art. In addition, forpurposes of the invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in “OrganicChemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999,and “Organic Chemistry”, Morrison & Boyd (3d Ed), the entire contents ofboth of which are incorporated herein by reference.

Step 1: (2-aminophenyl)dimethylphosphine oxide

A mixture of 2-iodoaniline (86 g, 0.393 mol, 1.0 eq.), dimethylphosphine oxide (36.4 g, 0.466 mol, 1.19 eq.), potassium phosphate (92.4g, 0.423 mol, 1.1 eq.), palladium(II) acetate (4.56 g, 0.02 mol, 0.05eq.), and Xantphos (11.6 g, 0.02 mol, 0.05 eq.) in DMF (700 mL) wasstirred at ˜120° C. for ˜6 h. The color of the mixture turned darkbrown. Upon cooling to rt, celite (30 g) was added to the mixture. Themixture was then filtered and the filter cake was rinsed with EtOAc(2×250 mL). The filtrate was then concentrated in vacuo to afford aresidue.

Another batch of (2-aminophenyl)dimethylphosphine oxide was synthesizedat the same scale as performed above, and the residue obtained from bothbatches were combined and purified as discussed below.

To the combined residues was added EtOAc (1 L), and the resultingmixture was stirred at rt for ˜1 h. The mixture was filtered, and thecollected residue was washed with EtOAc (2×250 mL). The combinedfiltrate was dried over sodium sulfate, filtered and concentrated invacuo to afford an oil. The resulting oil was dissolved in a mixture ofwater/concentrated hydrochloric acid (1.2 L/300 mL) with agitation atrt, and stirred for 30 min. The resulting mixture was filtered, and thecollected residue was washed with aqueous hydrochloric acid (10%, 300mL). The combined aqueous filtrate was washed with EtOAc (2×1 L washes,followed by a 500 mL wash). The aqueous layer was cooled in an ice bath(less than 10° C. internal mixture temperature) and the pH of thesolution was adjusted to ˜12 (as determined by pH paper) by addingaqueous sodium hydroxide (30% w/w), while maintaining an internalsolution temperature of less than 20° C. throughout the addition. Theresulting solution was extracted with IPA/DCM (⅓ v/v, 4×1 L), and thecombined organic layers were dried over sodium sulfate, filtered, andconcentrated in vacuo to afford a viscous oil, which crystallized uponstanding at rt. The resulting solids were triturated with EtOAc/heptane( 1/10 v/v, 2×150 mL) to afford (2-aminophenyl)dimethylphosphine oxideas a light brown solid.

Step 2: (2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)dimethylphosphineoxide

2,4,5-trichloropyrimidine (54.2 g, 0.296 mol, 1.0 eq.),(2-aminophenyl)dimethyl-phosphine oxide (50.0 g, 0.296 mole, 1.0 eq.),potassium carbonate (49.1 g, 0.355 mol, 1.2 eq.) and tetrabutylammoniumbisulfate (10.2 g, 0.03 mole, 0.1 eq.) were combined in DMF (1050 mL),and heated at 65° C. for ˜8.0-8.5 h. During the course of heating, anoff-white suspension formed. Upon cooling, the mixture was cooled to rtand filtered. The collected solids were rinsed with DMF (2×50 mL), andthe combined filtrates were concentrated in vacuo. The resulting residuewas dissolved in EtOAc (1.3 L) and water (350 mL). The aqueous layer wasisolated and extracted with EtOAc (2×250 mL). The combined organiclayers were washed with brine (20% w/w, 500 mL), dried over sodiumsulfate, filtered, and concentrated in vacuo to afford(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide asan off-white solid.

Alternative Synthesis of(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)-dimethylphosphine oxide

(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide canbe synthesized using the conditions in Table 28 according to thepreviously described procedure.

TABLE 28 Reaction Conditions for the Synthesis of(2-((2,5-dichloropyrimidin-4- yl)amino)phenyl)dimethylphosphine oxideAmount of (2- aminophenyl)- Amount of Phase dimethyl- 2,4,5- Transferphosphine trichloro- Base Catalyst Solvent(s), Entry oxide pyrimidine(equivalents) (mole %) Conditions 1 1.0 eq. 1.1 eq. K₂CO₃ N/A DMF (3eq.) 120° C., 6-8 h 2 1.0 eq. 1.1 eq. Cs₂CO₃ N/A Acetone (2.5 eq.)Reflux 3 1.0 eq. 1.2 eq. K₂CO₃ N/A Acetone (2.5 eq.) Reflux 4 1.0 eq.1.1 eq. Et₃N N/A MeCN (2.5 eq.) rt, then 80° C. for 6-8 h 5 1.0 eq. 1.1eq. Et₃N n-Bu₄I MeCN (2.5 eq.) (10 mole-%) 80° C., 6-8 h 6 1.0 eq. 1.1eq. KHCO₃ n-Bu₄I PhMe/H₂O (1/1, (1.2 eq.) (5 mole-%) v/v) rt to reflux 71.0 eq. 1.1 eq. KHCO₃ n-Bu₄I THF/H₂O (1/1, (1.2 eq.) (5 mole-%) v/v) rtto reflux 8 1.0 eq. 1.2 eq. KHCO₃ n-Bu₄I 2-Me-THF/H₂O (1.2 eq.) (5mole-%) (1/1, v/v) rt to reflux 9 1.0 eq. 1.1 eq. LiHMDS N/A −60° C. (2Msolution in THF, 2.1 eq.) 10 1.0 eq. 1.0 eq. K₂CO₃ n-Bu₄NHSO₄ 2-Me-THF(1.2 eq.) (10 mole-%) 65-70° C., 7-8 h 11 1.0 eq. 1.0 eq. K₂CO₃n-Bu₄NHSO₄ DMF (1.2 eq.) (10 mole-%) 60° C., 4-6 h

Step 3: 1-(1-(3-methoxy-4-nitrophenyl)piperidin-4-yl)-4-methylpiperazine

A mixture of 5-fluoro-2-nitroanisole (85.6 g, 0.5 mol, 1.0 eq.),1-methyl-4-(piperidin-4-yl)piperazine (91.7 g, 0.5 mol, 1.0 eq.), andpotassium carbonate (138.5 g, 1.0 mol, 2.0 eq.) in MeCN (500 mL) wasstirred at reflux for ˜13 h. Upon cooling to rt, DCM (1 L) was added tothe mixture and the resulting mixture was filtered. The collectedresidue was washed with DCM (500 mL). The combined filtrates were washedwith water (400 mL) and brine (20% w/w, 300 mL), dried over sodiumsulfate, filtered, and concentrated in vacuo to afford1-(1-(3-methoxy-4-nitrophenyl)piperidin-4-yl)-4-methylpiperazine as ayellow solid.

Step 4: 2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)aniline

A mixture of1-(1-(3-methoxy-4-nitrophenyl)piperidin-4-yl)-4-methylpiperazine (78 g,0.233 mol) and Pd/C (10% loading, 50% wet, 4 g, ˜2.5 wt-%) in EtOH (800mL) was stirred under a hydrogen atmosphere (˜20 p.s.i.) for ˜2.5 h.Subsequently, the mixture was filtered through a pad of Celite (50 g),and the Celite pad was rinsed with EtOH (2×50 mL). The combinedfiltrates were concentrated in vacuo to afford2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)aniline as apurple solid.

Step 5:(2-((5-chloro-2-((2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)phenyl)dimethylphosphineoxide

A mixture of(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)-dimethylphosphine oxide(55 g, 0.174 mol, 1.0 eq.),2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)aniline (74.2 g,0.244 mol, 1.4 eq.), and HCl in EtOH (2.5 M, 175 mL) in 2-methoxyethanol(750 mL) was stirred at 120° C. for ˜6 h. Upon cooling to rt, themixture was concentrated in vcauo, and the resulting residue wasdissolved in water (400 mL), and washed with EtOAc (500 mL). Aqueoussodium hydroxide (20% w/w) was added to the aqueous layer until the pHwas ˜12 (as determined by pH paper). The aqueous layer was extractedwith DCM (3×500 mL), and the combined organic layers were concentratedin vacuo. The residue was triturated with EtOAc/MeOH (9/1 v/v, 250 mL)and EtOAc/heptane (½ v/v, 300 mL), sequentially, at rt for ˜1 h, andthen filtered to afford a light color solid (Batch A).

Another batch of(2-((5-chloro-2-((2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)phenyl)dimethyl-phosphineoxide was prepared using(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide(50.8 g, 0.161 mol, 1.0 eq.),2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)aniline (68.4 g,0.225 mol, 1.4 eq.), and HCl in EtOH (2.5 M, 160 mL) in 2-methoxyethanol(650 mL). After the previously described workup, a solid was obtained(Batch B).

The two batches (Batch A and Batch B) were combined and triturated withMeOH/EtOAc (1% v/v, 500 mL) and MeOH/EtOAc (2.5% v/v, 500 mL) at rt for−30 min, and then filtered. The isolated solids were then trituratedwith hot EtOAc (500 mL) for 15 minutes followed by cooling to rt, andthen filtration. The isolated solids were then triturated in hotMeOH/EtOAc (2% v/v, 500 mL) for 15 minutes followed by cooling to roomtemperature and filtration. Then the isolated solids were triturated inDCM (750 mL) at room temperature. The resulting solution was filteredand the collected solid was dried in vacuo to afford(2-((5-chloro-2-((2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)phenyl)dimethylphosphineoxide as a beige solid. 127 g, 65% yield. ¹H NMR: refer to Table 2.ESI-MS m/s: 584.2 [M+H]⁺.

VI. Pharmaceutical Composition Examples

Representative pharmaceutical compositions and dosage forms of compoundsas disclosed herein (the active ingredient being referred to as“Compound”) for therapeutic or prophylactic use in humans may be asfollows:

(a) Tablet I mg/tablet Compound 100 Lactose Ph.Eur 182.75 Croscarmellosesodium 12.0 Maize starch paste (5% w/v paste) 2.25 Magnesium stearate3.0 (b) Tablet II mg/tablet Compound 50 Lactose Ph.Eur 223.75Croscarmellose sodium 6.0 Maize starch 15.0 Polyvinylpyffolidone (5% w/vpaste) 2.25 Magnesium stearate 3.0 (c) Tablet III mg/tablet Compound 1.0Lactose Ph.Eur 93.25 Croscarmellose sodium 4.0 Maize starch paste (5%w/v paste) 0.75 Magnesium stearate 1.0-76  (d) Capsule mg/capsuleCompound 10 Lactose Ph.Eur 488.5 Magnesium 1.5 (e) Injection I (50mg/mL) Compound  5.0% w/v 1M Sodium hydroxide solution 15.0% v/v 0. 1MHydrochloric acid (to adjust pH to 7.6) Polyethylene glycol 400  4.5%w/v Water for injection to 100% (f) Injection II (10 mg/mL) Compound 1.0% w/v Sodium phosphate BP  3.6% w/v 0. 1M Sodium hydroxide solution15.0% v/v Water for injection to 100% (g) Injection III (1 mg/mL,buffered to pH 6) Compound  0.1% w/v Sodium phosphate BP 2.26% w/vCitric acid 0.38% w/v Polyethylene glycol 400  3.5% w/v Water forinjection to 100% (h) Aerosol I mg/mL Compound 10.0 Sorbitan trioleate13.5 Trichlorofluoromethane 910.0 Dichlorodifluorometha-ne 490.0 (i)Aerosol II mg/mL Compound 0.2 Sorbitan trioleate 0.27Trichlorofluoromethane 70.0 Dichlorodifluoromethane 280.0Dichlorotetrafluoroethane 1094.0 (j) Aerosol III mg/mL Compound 2.5Sorbitan trioleate 3.38 Trichlorofluoromethane 67.5Dichlorodifluoromethane 1086.0 Dichlorotetrafluoroethane 191.6 (k)Aerosol IV mg/mL Compound 2.5 Soya lecithin 2.7 Trichlorofluoromethane67.5 Dichlorodifluoromethane 1086.0 Dichlorotetrafluoroethane 191.6 (l)Ointment unit/mL Compound  40 mg Ethanol 300 μL Water 300 μL1-Dodecylazacycloheptan one  50 μL Propylene glycol to 1 mL

These formulations can be prepared using conventional procedures wellknown in the pharmaceutical art. The tablets (a)-(c) can be entericcoated by conventional means, if desired to provide a coating ofcellulose acetate phthalate, for example. In certain embodiments,tablets suitable for oral administration contain about 30 mg, about 90mg, about 150 mg, or about 180 mg of substantially pure Form A ofbrigatinib, together with one or more pharmaceutically acceptableexcipients such as are described herein. As used herein, “about” means±5% of the value being modified. The aerosol formulations (h)-(k) can beused in conjunction with standard, metered dose aerosol dispensers, andthe suspending agents sorbitan trioleate and soya lecithin can bereplaced by an alternative suspending agent such as sorbitan monooleate,sorbitan sesquioleate, polysorbate 80, polyglycerol oleate or oleicacid.

VII. Kinase Inhibition

Compounds as described herein were screened for kinase inhibitionactivity as follows. Kinases suitable for use in the following protocolinclude, but are not limited to: ALK, Jak2, b-Raf, c-Met, Tie-2, FLT3,Abl, Lck, Lyn, Src, Fyn, Syk, Zap-70, Itk, Tec, Btk, EGFR, ErbB2, Kdr,FLT1, Tek, InsR, and AKT.

Kinases are expressed as either kinase domains or full length constructsfused to glutathione S-transferase (GST) or polyHistidine tagged fusionproteins in either E. coli or Baculovirus-High Five expression systems.They are purified to near homogeneity by affinity chromatography aspreviously described (Lehr et al., 1996; Gish et al., 1995). In someinstances, kinases are co-expressed or mixed with purified or partiallypurified regulatory polypeptides prior to measurement of activity.

Kinase activity and inhibition can be measured by established protocols(see e.g., Braunwalder et al., 1996). In such cases, the transfer of³³PO₄ from ATP to the synthetic substrates poly(Glu, Tyr) 4:1 orpoly(Arg, Ser) 3:1 attached to the bioactive surface of microtiterplates is taken as a measure of enzyme activity. After an incubationperiod, the amount of phosphate transferred is measured by first washingthe plate with 0.5% phosphoric acid, adding liquid scintillant, and thencounting in a liquid scintillation detector. The IC₅₀ is determined bythe concentration of compound that causes a 50% reduction in the amountof ³³P incorporated onto the substrate bound to the plate.

Other methods relying upon the transfer of phosphate to peptide orpolypeptide substrate containing tyrosine, serine, threonine orhistidine, alone, in combination with each other, or in combination withother amino acids, in solution or immobilized (i.e., solid phase) arealso useful.

For example, transfer of phosphate to a peptide or polypeptide can alsobe detected using scintillation proximity, Fluorescence Polarization andhomogeneous time-resolved fluorescence. Alternatively, kinase activitycan be measured using antibody-based methods in which an antibody orpolypeptide can used as a reagent to detect phosphorylated targetpolypeptide.

For additional background information on such assay methodologies, seee.g., Braunwalder et al., 1996, Anal. Biochem. 234(1):23; Cleaveland etal., 1990, Anal Biochem. 190(2):249; Gish et al. (1995). Protein Eng.8(6):609; Kolb et al. (1998). Drug Discov. Toda V. 3:333; Lehr et al.(1996). Gene 169(2):27527-87; Seethala et al. (1998). Anal Biochem.255(2):257; Wu et al. (2000).

The inhibition of ALK tyrosine kinase activity can be demonstrated usingknown methods. For example, in one method, compounds can be tested fortheir ability to inhibit kinase activity of baculovirus-expressed ALKusing a modification of the ELISA protocol reported for trkA in Angeles,T. S. et al., Anal. Biochem. 1996, 236, 49-55, which is incorporatedherein by reference. Phosphorylation of the substrate, phopholipaseC-gamma (PLC-γ) generated as a fusion protein withglutathione-S-transferase (GST) as reported in rotin, D. et al., EMBO J.1992, 11, 559-567, which is incorporated by reference, can be detectedwith europium-labeled anti-phosphotyrosine antibody and measured bytime-resolved fluorescence (TRF). In this assay, 96-well plate is coatedwith 100 μL/well of 10 μg/mL substrate (phospholipase C-γ intris-buffered saline (TBS). The assay mixture (total volume=100 μL/well)consisting of 20 nM HEPES (pH 7.2, 1 μM ATP (K_(m) level), 5 nM MnCl₂,0.1% BSA, 2.5% DMSO, and various concentrations of test compound is thenadded to the assay plate. The reaction is initiated by adding the enzyme(30 ng/mL ALK) and is allowed to proceed at 37 degrees C. for 15minutes. Detection of the phosphorylated product can be performed byadding 100 μL/well of Eu—N1 labeled PT66 antibody (Perkim Elmer#AD0041). Incubation at 37 degrees C. then proceeds for one hour,followed by addition of 100 μL enhancement solution (for example Wallac#1244-105). The plate is gently agitated and after thirty minutes, thefluorescence of the resulting solution can be measured (for exampleusing EnVision 2100 (or 2102) multilabel plate reader from PerkinElmer).

Data analysis can then be performed. IC₅₀ values can be calculated byplotting percent inhibition versus log₁₀ of concentration of compound.

The inhibition of ALK tyrosine kinase activity can also be measuredusing the recombinant kinase domain of the ALK in analogy to VEDG-Rkinase assay described in J. Wood et al., Cancer Res 2000, 60,2178-2189. In vitro enzyme assays using GST-ALK protein tyrosine kinasecan be performed in 96-well plate as a filter binding assay in 20 mMTris.HCl, pH 7.5, 3 mM MgCl₂, 10 mM MnCl₂, 1 nM DTT, 0.1 μCi/assay (=30μL) [γ-³³P]-ATP, 2 μM ATP, 3 μg/mL poly (Glu, tyr 4:1) Poly-EY (sigmaP-0275), 1% DMSO, 25 ng ALK enzyme. Assays can be incubated for 10 min,at ambient temperature. Reactions can be terminated by adding 50 μL of125 mM EDTA, and the reaction mixture can be transferred onto a MAIPMultiscreen plate (Millipore, Bedford, Mass.) previously wet withmethanol, and rehydrated for 5 minutes with water. Following washing(0.5% H₃PO₄), plates can be counted in a liquid scintillation counter.IC₅₀ values are calculated by linear regression analysis of thepercentage inhibition.

Certain compounds as disclosed herein have also been demonstratedcytotoxic or growth inhibitory effects on tumor and other cancer celllines and thus can be useful in the treatment of cancer and other cellproliferative diseases. Compounds are assayed for anti-tumor activityusing in vivo and in vitro assays which are well known to those skilledin the art. Generally, initial screens of compounds to identifycandidate anti-cancer drugs are performed in cellular assays. Compoundsidentified as having antiproliferative activity in such cell-basedassays can then be subsequently assayed in whole organisms foranti-tumor activity and toxicity. Generally speaking, cell-based screenscan be performed more rapidly and cost-effectively relative to assaysthat use whole organisms. As disclosed herein, the terms “anti-tumor”and “anti-cancer” activity are used interchangeably.

Cell-based methods for measuring antiproliferative activity are wellknown and can be used for comparative characterization of compounds asdisclosed herein. In general, cell proliferation and cell viabilityassays are designed to provide a detectable signal when cells aremetabolically active. Compounds can be tested for antiproliferativeactivity by measuring any observed decrease in metabolic activity of thecells after exposure of the cells to compound. Commonly used methodsinclude, for example, measurement of membrane integrity (as a measure ofcell viability)(e.g. using trypan blue exclusion) or measurement of DNAsynthesis (e.g. by measuring incorporation of BrdU or 3H-thymidine).

Some methods for assaying cell proliferation use a reagent that isconverted into a detectable compound during cell proliferation. Suchreagents are tetrazolium salts and include without limitation MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma-Aldrich,St. Louis, Mo.), MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium),XTT(2,3-bis(2-Methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide),INT, NBT, and NTV (Bernas et al. Biochim Biophys Acta 1451(1):73-81,1999). More commonly used assays utilizing tetrazolium salts detect cellproliferation by detecting the product of the enzymatic conversion ofthe tetrazolium salts into blue formazan derivatives, which are readilydetected by spectroscopic methods (Mosman. J. Immunol. Methods.65:55-63, 1983).

Other methods for assaying cell proliferation involve incubating cellsin a given growth medium with and without the compounds to be tested.Growth conditions for various prokaryotic and eukaryotic cells arewell-known to those of ordinary skill in the art (Ausubel et al. CurrentProtocols in Molecular Biology. Wiley and Sons. 1999; Bonifacino et al.Current Protocols in Cell Biology. Wiley and Sons. 1999, bothincorporated herein by reference). To detect cell proliferation, thetetrazolium salts are added to the incubated cultured cells to allowenzymatic conversion to the detectable product by active cells. Cellsare processed, and the optical density of the cells is determined tomeasure the amount of formazan derivatives. Furthermore, commerciallyavailable kits, including reagents and protocols, are availabe forexamples, from Promega Corporation (Madison, Wis.), Sigma-Aldrich (St.Louis, Mo.), and Trevigen (Gaithersburg, Md.).

In addition, a wide variety of cell types can be used to screencompounds for antiproliferative activity, including the following celllines, among others: COLO 205 (colon cancer), DLD-1 (colon cancer),HCT-15 (colon cancer), HT29 (colon cancer), HEP G2 (Hepatoma), K-562(Leukemia), A549 (Lung), NCI-H249 (Lung), MCF7 (Mammary), MDA-MB-231(Mammary), SAOS-2 (Osteosarcoma), OVCAR-3 (Ovarian), PANC-1 (Pancreas),DU-145 (Prostate), PC-3 (Prostate), ACHN (Renal), CAKI-1 (Renal), MG-63(Sarcoma).

While the cell line is can be mammalian, lower order eukaryotic cellssuch as yeast can also be used to screen compounds. Mammalian cell linesare derived from humans, rats, mice, rabbits, monkeys, hamsters, andguinea pigs since cells lines from these organisms are well-studied andcharacterized. However, others can be used as well.

Suitable mammalian cell lines are often derived from tumors. Forexample, the following tumor cell-types can be sources of cells forculturing cells: melanoma, myeloid leukemia, carcinomas of the lung,breast, ovaries, colon, kidney, prostate, pancreas and testes),cardiomyocytes, endothelial cells, epithelial cells, lymphocytes (T-celland B cell), mast cells, eosinophils, vascular intimal cells,hepatocytes, leukocytes including mononuclear leukocytes, stem cellssuch as haemopoetic, neural, skin, lung, kidney, liver and myocyte stemcells (for use in screening for differentiation and de-differentiationfactors), osteoclasts, chondrocytes and other connective tissue cells,keratinocytes, melanocytes, liver cells, kidney cells, and adipocytes.Non-limiting examples of mammalian cells lines that have been widelyused by researchers include HeLa, NIH/3T3, HT1080, CHO, COS-1, 293T,WI-38 and CV1/EBNA-1.

Other cellular assays can be used which rely upon a reporter gene todetect metabolically active cells. Non-limiting examples of reportergene expression systems include green fluorescent protein (GFP), andluciferase. As an example of the use of GFP to screen for potentialantitumor drugs, Sandman et al. (Chem Biol. 6:541-51; incorporatedherein by reference) used HeLa cells containing an inducible variant ofGFP to detect compounds that inhibited expression of the GFP, and thusinhibited cell proliferation.

An example of a cell-based assay is shown below. The cell lines that canbe used in the assay are Ba/F3, a murine pro-B cell line, which has beenstably transfected with an expression vector pCIneo™ (Promega Corp.,Madison Wis.) coding for NPM-ALK and subsequent selection of G418resistant cells. Non-transfected Ba/F3 cells depend on IL-3 for cellsurvival. In constrast NPM-ALK expressing Ba/F3 cells (namedBa/F3-NPM-ALK) can proliferate in the absence of IL-3 because theyobtain proliferative signal through NPM-ALK kinase. Putative inhibitorsof NPM-ALK kinase therefore abolish the growth signal and result inantiproliferative activity. The antiproliferative activity of inhibitorsof the NPM-ALK kinase can however be overcome by addition of IL-3 whichprovides growth signals through an NPM-ALK independent mechanism. For ananalogous cell system using FLT3 kinase, see E. Weisberg et al. Cancercell, 2002, 1, 433-443. The inhibitory activity of compounds asdisclosed herein can be determined as follows: BaF3-NPM-ALK cells(15,000/microtitre plate well) can be transferred to a 96-wellmicrotitre plates. The test compound (dissolved in DMSO) is then addedin a series of concentrations (dilution series) in such a manner thatthe final concentration of DMSO is not greater than 1% (v/v). After theaddition, the plates can be incubated for two days during which thecontrol cultures without test compound are able to undergo twocell-division cycles. The growth of BaF3-NPM-ALK cells can be measuredby means of Yopro™ staining (T Idziorek et al., J. Immunol. Methods1995, 185, 249-258). Then, 25 μL of lysis buffer consisting of 20 mMsodium citrate, pH 4.0, 26.8 nM sodium chloride, 0.4% NP40, 20 mM EDTAand 20 mM is added into each well. Cell lysis is completed within 60minutes at room temperature and total amount of Yopro bound to DNA isdetermined by measurement using for example a CytoFluor II 96-wellreader (PerSeptive Biosystems). The IC₅₀ can be determined by a computeraided system using the formula:IC₅₀=[(ABS_(test)−ABS_(start))/(ABS_(control)−ABS_(start))]×100in which ABS is absorption. The IC₅₀ value in such an experiment isgiven as that concentration of the test compound in question thatresults in a cell count that is 50% lower than that obtained using thecontrol without inhibitor.

The antiproliferative action of compounds as disclosed herein can alsobe determined in the human KARPAS-299 lymphoma cell line by means of animmunoblot as described in W G Dirks et al. Int. J. Cancer 2002, 100,49-56., using the methodology described above for the BaF3-NPM-ALK cellline.

In another example, antiproliferative activity can be determined usingKARPAS-299 lumphoma cell line in the following procedure: Compounds asdisclosed herein were incubated with the cells for 3 days, and thenumber of viable cells in each well was measured indirectly using an MTStetrazolium assay (Promega). This assay is a colorimetric method fordetermining the number of viable cells through measurement of theirmetabolic activity. For example the detection of the product of theenzymatic conversion of tetrazolium salts into blue formazan derivativesis achieved by measuring absorbance at 490 nm using a plate reader. 40μL of the MTS reagent was added to all wells except the edge wells andthen the plates were returned to the incubator at 37° C. for 2 hours.The absorbance in each well was then measured at 490 nm using a WallacVictor²V plate reader. The IC₅₀ was calculated by determining theconcentration of compound required to decrease the MTS signal by 50% inbest-fit curves using Microsoft XLfit software, by comparing withbaseline, the DMSO control, as 0% inhibition.

Compounds identified by such cellular assays as having anti-cellproliferation activity can then be tested for anti-tumor activity inwhole organisms, such as mammalian species. Well-characterizedmammalians systems for studying cancer include rodents such as rats andmice. Typically, a tumor of interest is transplanted into a mouse havinga reduced ability to mount an immune response to the tumor to reduce thelikelihood of rejection. Such mice include for example, nude mice(athymic) and SCID (severe combined immunodeficiency) mice. Othertransgenic mice such as oncogene containing mice can be used in thepresent assays (see for example U.S. Pat. Nos. 4,736,866 and 5,175,383).For a review and discussion on the use of rodent models for antitumordrug testing, see Kerbel (Cancer Metastasis Rev. 17:301-304, 1998-99).

In general, the tumors of interest are implanted in a test organismsubcutaneously. The organism containing the tumor is treated with dosesof candidate anti-tumor compounds. The size of the tumor is periodicallymeasured to determine the effects of the test compound on the tumor.Some tumor types are implanted at sites other than subcutaneous sites(e.g. intraperitoneal sites) and survival is measured as the endpoint.Parameters to be assayed with routine screening include different tumormodels, various tumor and drug routes, and dose amounts and schedule.For a review of the use of mice in detecting antitumor compounds, seeCorbett et al. (Invest New Drugs. 15:207-218, 1997; incorporated hereinby reference).

The compounds disclosed herein have inhibitory activity against a wildtype or mutant (especially a clinically relevant mutant) kinase,especially a kinase such as ALK, Met, Jak2, bRaf, EGFR, Tie-2, FLT3 oranother kinase of interest with an IC₅₀ value of 1 μM or less (asdetermined using any scientifically acceptable kinase inhibition assay),such as with an IC₅₀ of 500 nM or better, and further such as an IC₅₀value of 250 nM or better; or

-   -   inhibitory activity against a given kinase with an IC₅₀ value at        least 100-fold lower than their IC₅₀ values for other kinases of        interest; or    -   inhibitory activity for ALK, Met, Jak2 or B-Raf with a 1 μM or        better IC₅₀ value against each; or    -   a cytotoxic or growth inhibitory effect on cancer cell lines        maintained in vitro, or in animal studies using a scientifically        acceptable cancer cell xenograft model, (such as Ba/F3 NPM-ALK,        Ba/F3 EML4-ALK, Karpas 299 and/or SU-DHL-1 cells with a potency        at least as great as the potency of known ALK inhibitors such as        NVP-TAE684 and PF2341066 among others, or with a potency at        least twice that of known ALK inhibitors, or with a potency at        least 10 times that of known ALK inhibitors as determined by        comparative studies.

Compounds disclosed herein were found to potently inhibit a number ofimportant kinase targets. Compounds exhibited IC₅₀'s under 100 nM, andin many cases under 10 nM and in some cases under 1 nM when tested asinhibitors of the kinase, ALK, for instance. Some compounds were singledigit nanomolar inhibitors of a panel of kinases including kinases likeALK, FER, FLT3, FES/FPS, FAK/PTK2, BRK and others.

What is claimed is:
 1. Crystalline Form H of brigatinib having thestructure:

wherein the crystalline form has an x-ray powder diffraction patternwith peaks at 4.2, 5.2, 8.4, 10.9, 12.7, and 21.3° 2θ, with a varianceof ±0.2° 2θ.
 2. The crystalline Form H of claim 1 having an x-ray powderdiffraction pattern as shown in FIG.
 25. 3. The crystalline Form H ofclaim 1 having an x-ray powder diffraction pattern with characteristicpeaks at 4.2, 5.2, 8.4, 10.9, 12.7, 15.0, 15.7, 16.5, 17.2, 18.4, 19.5,and 21.3° 2θ, with a variance of ±0.2° 2θ.
 4. A pharmaceuticalcomposition comprising the crystalline Form H of brigatinib of claim 1and at least component chosen from pharmaceutically acceptable carriers,pharmaceutically acceptable vehicles, and pharmaceutically acceptableexcipients.
 5. A method for treating non-small cell lung cancer in asubject in need thereof comprising administering to the subject thecrystalline Form H of brigatinib of claim
 1. 6. The method of claim 5,wherein the non-small cell lung cancer is ALK-positive non-small celllung cancer.
 7. The method of claim 6, wherein the non-small cell lungcancer is ALK-positive metastatic non-small cell lung cancer.
 8. Themethod of claim 7, wherein the subject has been previously treated withcrizotinib.